Methods for preventing or treating cardiovascular conditions using il-22 fc fusion proteins

ABSTRACT

The invention relates to IL-22 polypeptides, IL-22 Fc fusion proteins and IL-22 agonists, composition comprising the same, methods of making and methods of using the composition for the treatment of diseases. The invention also relates to IL-22 receptor associated reagents and methods of use thereof.

The instant application is a divisional of U.S. application Ser. No.14/214,161, filed Mar. 14, 2014, now U.S. Pat. No. 10,160,793, whichclaims the benefit of priority to U.S. provisional application Ser. Nos.61/800,148, 61/800,795 and 61/801,144, all of which were filed on Mar.15, 2013, U.S. provisional application Ser. No. 61/821,062, filed on May8, 2013, and U.S. provisional application Ser. No. 61/860,176, filed onJul. 30, 2013, the contents of all of which are herein incorporated byreference in their entirety.

SEQUENCE LISTING

The instant application contains a Sequence Listing submitted viaEFS-Web and hereby incorporated by reference in its entirety. Said ASCIIcopy, created on Nov. 16, 2018, is named50474-1350030_Sequence_Listing_11.16.18_ST25.txt, and is 106,782 bytesin size.

FIELD

The present invention relates to IL-22 and IL-22 Fc fusion proteins,IL-22 agonists, compositions comprising the same, and methods of makingand method of using the same.

BACKGROUND

Interleukin-22 (IL-22) is a member of the IL-10 family of cytokine thatis produced by Th22 cells, NK cells, lymphoid tissue inducer (LTi)cells, dendritic cells and Th17 cells. IL-22 binds to theIL-22R1/IL-10R2 receptor complex, which is expressed in innate cellssuch as epithelial cells, hepatocytes, and keratinocytes and in barrierepithelial tissues of several organs including dermis, pancreas,intestine and the respiratory system.

IL-22 plays an important role in mucosal immunity, mediating early hostdefense against attaching and effacing bacterial pathogens. See Zheng etal., 2008, Nat. Med. 14:282-89. IL-22 promotes the production ofanti-microbial peptides and proinflammatory cytokines from epithelialcells and stimulates proliferation and migration of colonic epithelialcells in the gut. See Kumar et al., 2013, J. Cancer, 4:57-65. Uponbacterial infection, IL-22 knock-out mice displayed impaired gutepithelial regeneration, high bacterial load and increased mortality.Kumar et al., supra. Similarly, infection of IL-22 knock-out mice withinfluenza virus resulted in severe weight loss and impaired regenerationof tracheal and bronchial epithelial cells. Thus, IL-22 plays apro-inflammatory role in suppressing microbial infection as well as ananti-inflammatory protective role in epithelial regeneration ininflammatory responses. Much of IL-22's biological action promotingpathological inflammation and tissue repair remains to be determined.The seemingly conflicting reports on the effects of IL-22 on epithelialcells are not yet thoroughly understood. Kumar et al., supra.

The regulation of antimicrobial defensins, which limits bacterialreplication and dissemination, would help to stabilize intestinalmicrobiota by reducing subsequent LPS production, and preserving mucosalintegrity. IL-22 up-regulates expression of acute phase proteins,including SAA, and contributes to the expression of a range of genesassociated with acute inflammatory responses, including IL-6, G-CSF, andIL-1a. Systemic administration of IL-22 to healthy mice also upregulates LPS binding proteins to physiologically relevantconcentrations for neutralizing LPS in response to bacterial infection.

Increased expression of IL-22 is detected in inflammatory bowel disorder(IBD) patients. See e.g., Wolk et al., 2007, J. Immunology, 178:5973;Andoh et al., 2005, Gastroenterology, 129:969. IBDs such as Crohn'sdisease (CD) and ulcerative colitis (UC) are thought to result from adysregulated immune response to the commensal microflora present in thegut. Cox et al., 2012, Mucosal Immunol. 5:99-109. Both UC and CD arecomplex diseases that occur in genetically susceptible individuals whoare exposed to as yet poorly-defined environmental stimuli. CD and UCare mediated by both common and distinct mechanisms and exhibit distinctclinical features. See Sugimoto et al. 2008, J. Clinical Investigation,118:534-544.

In UC, inflammation occurs primarily in the mucosa of the colon and therectum, leading to debilitating conditions including diarrhea, rectalbleeding, and weight loss. It is thought that UC is largely caused by aninappropriate inflammatory response by the host to intestinal microbespenetrating through a damaged epithelial barrier (Xavier and Podolsky,2007, Nature 448:427-434). Crohn's disease is characterized byintestinal infiltration of activated immune cells and distortion of theintestinal architechture. See Wolk et al., supra.

In recent years, a number of drugs based on various strategies toregulate the immune response have been tested to treat IBD, includingsteroids, immunomodulators, and antibodies against inflammatorycytokines, with variable success (Pastorelli et al., Expert opinion onemerging drugs, 2009, 14:505-521). The complex variety of gut floracontributes to the heterogeneity of the disease. Thus, there is a needfor a better therapeutics for IBD.

Cardiovascular disease (CVD) is a leading cause of mortality thatresults, in part, from atherosclerotic disease of large blood vessels.Atherosclerosis is the major culprit in CVD events and is a slow andprogressive disease that results from hypercholesterolemia andchronically inflamed blood vessels. Atherosclerotic lesions arecharacterized as lipid laden with infiltration of immunocytes,especially macrophages and T cells. It is now acknowledged that both theinnate and adaptive immune mechanisms contribute to the progression andeventual thrombosis of the atherogenic plaque (Ross, Am Heart J. 1999November; 138 (5 Pt 2):S419-20; Hansson 2005 N Engl J Med 352(16):1685-95; Hansson and Hermansson 2011 Nature Immunology 12(3): 204-12).

Acute pancreatitis (AP) is an acute inflammatory process of thepancreas. Acute kidney injury (AKI) is an abrupt loss of kidneyfunction, resulting in the retention of urea and other nitrogenous wasteproducts and in the dysregulation of extracellular volume andelectrolytes. AKI was previously known as acute kidney failure. Thechange reflects recent recognition that even smaller decreases in kidneyfunction that do not result in overt organ failure are of substantialclinical relevance and are associated with increased morbidity andmortality. There remains a need for better treatment for AP and AKI.

Metabolic syndrome is a complex state characterized by a series of riskfactors that contribute to thrombosis, hypertension, dyslipidemia, andinflammation. Insulin resistance and obesity are major pathogenicmechanisms underlying the metabolic syndrome.

Insulin resistance increases CVD risk because it induces endothelialdysfunction which, in combination with atherogenic dyslipidemia,inflammation, and hypertension, contributes to the mortality fromcoronary artery disease (CAD). Persistent insulin resistance alsoincreases the chance of developing diabetes mellitus type 2 (T2DM)although the atherogenic state occurs many years before the onset ofT2DM. It is likely therefore that the natural history of CAD lies in thesame pathway as T2DM but begins much earlier in life in a subclinicalform, taking longer to manifest clinically, with or without the presenceof diabetes.

The term metabolic endotoxemia was coined to describe the condition ofincreased plasma LPS induced by, for example, high-fat high-calorie diet(HFD) (Cani et al. 2007. Diabetes 56(7): 1761-72). Mice fed with HFDhave increased plasma levels of bacterial lipopolysaccharide (LPS) andthis elevation appears to be a direct consequence of the increaseddietary fat (Cani et al. 2007 supra; Cani et al. 2008 Diabetes 57(6):1470-81; Ghoshal et al. 2009, J Lipid Res 50(1): 90-7). There iscompelling evidence that gut microbiota play an integral part in thehost's energy balance and harvest of dietary nutrients and carbohydratemetabolism, through modulation of gut mucosal epithelial cell function(Turnbaugh et al. 2009, J Physiol (Lond) 587(Pt 17): 4153-8; Manco etal. 2010, Endocr Rev 31(6): 817-44). Alteration in gut microbiota thatoccurs through disproportionate dietary fat composition or excessdietary caloric consumption is a recognized initiator of obesity andinsulin resistance, the established sequela of cardiovascular disease.Lipopolysaccharides are found in outer membrane of gram-negativebacteria and act as a source of endotoxin that can elicit a strongimmune response (Barcia et al. Clin Infect Dis 41 Suppl 7: S498-503).Alterations in the population, species and regional distribution ofintestinal microbiota can lead to changes in catabolism of LPS and ahigh fat diet will facilities adsorption of LPS across the intestinalbarrier. Under these conditions, increased LPS in systemic circulationwill induce low grade chronic inflammation, activating the endogenousprotective host response to elevate plasma lipids that, in the chroniccondition, contributes to diet induced obesity, insulin resistance andatherosclerosis, and eventual CVD events.

Diabetes mellitus is a serious metabolic disease that is defined by thepresence of chronically elevated levels of blood glucose(hyperglycemia). This state of hyperglycemia is the result of a relativeor absolute lack of activity of the peptide hormone, insulin. Insulin isproduced and secreted by the β cells of the pancreas. Insulin isreported to promote glucose utilization, protein synthesis, and theformation and storage of carbohydrate energy as glycogen. Glucose isstored in the body as glycogen, a form of polymerized glucose, which canbe converted back into glucose to meet metabolism requirements. Undernormal conditions, insulin is secreted at both a basal rate and atenhanced rates following glucose stimulation, all to maintain metabolichomeostasis by the conversion of glucose into glycogen. There remains aneed for new treatment paradigms for atherosclerosis and prevention ofCVD events, metabolic syndrome, acute endotoxemia and sepsis, andinsulin-related disorders.

Wound healing is a complex process, involving an inflammation phase, agranulation tissue formation phase, and a tissue remodeling phase (see,e.g., Singer and Clark, Cutaneous Wound Healing, N. Engl. J. Med.341:738-46 (1999)). These events are triggered by cytokines and growthfactors that are released at the site of injury. Many factors cancomplicate or interfere with normal adequate wound healing. For example,such factors include age, infection, poor nutrition, immunosuppression,medications, radiation, diabetes, peripheral vascular disease, systemicillness, smoking, and stress.

For subjects with diabetes, a chronic, debilitating disease, developmentof a diabetic foot ulcer (also referred to as a wound) is a commoncomplication. A chronic ulcer is defined as a wound that does notproceed through an orderly and timely repair process to produce anatomicand functional integrity (see, e.g., Lazarus et al., Definitions andguidelines for assessment of wounds and evaluation of healing, Arch.Dermatol. 130:489-93 (1994)). By its nature, the diabetic foot ulcer isa chronic wound (American Diabetes Association, Consensus developmentconference on diabetic foot wound care, Diabetes Care, 22(8):1354-60(1999)). Because the skin serves as the primary barrier again theenvironment, an open refractory wound can be catastrophic; a majordisability (including limb loss) and even death can result. Footulceration is the precursor to about 85% of lower extremity amputationsin persons with diabetes (see, e.g., Apelqvist, et al., What is the mosteffective way to reduce incidence of amputation in the diabetic foot?Diabetes Metab Res. Rev., 16(1 Suppl.): S75-S83 (2000)). Thus, there isa need for accelerating or improving wound healing, including diabeticwound healing.

SUMMARY

In one aspect, the invention provides IL-22 Fc fusion proteins,compositions comprising the same, and methods of using the same.

In one aspect, the invention provides an IL-22 Fc fusion protein thatbinds to IL-22 receptor, said IL-22 Fc fusion protein comprising anIL-22 polypeptide linked to an Fc region by a linker, wherein the Fcregion comprises a hinge region, an IgG CH2 domain and an IgG CH3domain, wherein the IL-22 Fc fusion protein comprises an amino acidsequence having at least 95%, at least 96%, at least 97%, at least 98%,preferably at least 99% sequence identity to the amino acid sequenceselected from the group consisting of SEQ ID NO:8, SEQ ID NO:10, SEQ IDNO:12 and SEQ ID NO:14, and wherein the Fc region is not glycosylated.In certain embodiments, the N297 residue of the CH2 domain is changed toglycine or alanine. In certain other embodiments, the N297 residue ischanged to Gly; while in other embodiments, the N297 residue is changedto Ala. In certain embodiments, the binding to IL-22 receptor triggersIL-22 receptor downstream signaling, including activating STAT3.

In certain embodiments, the IL-22 Fc fusion protein comprises an aminoacid sequence having at least 98% sequence identity to the amino acidsequence of SEQ ID NO:8 or SEQ ID NO:12. In certain other embodiments,the IL-22 Fc fusion protein comprises an amino acid sequence having atleast 99% sequence identity to the amino acid sequence of SEQ ID NO:8 orSEQ ID NO:12. In certain other embodiments, the IL-22 Fc fusion proteincomprises an amino acid sequence having at least 99% sequence identityto the amino acid sequence of SEQ ID NO:8. In certain other embodiments,the IL-22 Fc fusion protein comprises an amino acid sequence having atleast 99% sequence identity to the amino acid sequence of SEQ ID NO:12.In certain embodiments, the functions and/or activities of the IL-22 Fcfusion protein can be assayed by in vitro or in vivo methods, forexample, IL-22 receptor binding assay, Stat3 luciferase reporteractivity assay, etc. In certain embodiments, the IL-22 Fc fusion proteincomprises the amino acid sequence of SEQ ID NO:8 or SEQ ID NO:12. Incertain particular embodiments, the IL-22 Fc fusion protein comprisesthe amino acid sequence of SEQ ID NO:8. In certain embodiments, theinvention provides the IL-22 Fc fusion protein produced by the methodcomprising the step of culturing a host cell capable of expressing theIL-22 Fc fusion protein under conditions suitable for expression of theIL-22 Fc fusion protein. In certain embodiments, the method furthercomprises the step of obtaining the IL-22 Fc fusion protein from thecell culture or culture medium. In certain embodiments, the host cell isa Chinese hamster ovary (CHO) cell; while in other embodiments, the hostcell is an E. coli cell.

In another aspect, the invention provides an IL-22 Fc fusion proteincomprising an IL-22 polypeptide linked to an IgG Fc region by a linker,wherein the Fc region comprises a hinge region, an IgG CH2 domain and anIgG CH3 domain, and wherein the Fc region is not glycosylated. Incertain embodiments, the hinge region comprises the amino acid sequenceof CPPCP (SEQ ID NO:31). In certain other embodiments, the N297 residuein the Fc region is changed and/or the T299 residue in the Fc region ischanged. In certain embodiments, the N297 residue in the CH2 domain ischanged, preferably to glycine or alanine. In certain particularembodiments, the N297 residue is changed to glycine. In certain otherembodiments, the N297 residue is changed to alanine. In yet otherembodiments, the T299 residue is changed to Ala, Gly or Val. In certainother embodiments, the linker is 8-20 amino acids long, 8-16 amino acidslong, or 10-16 amino acids long.

In certain embodiments, the Fc region comprises the CH2 and CH3 domainof IgG1. In certain particular embodiments, the linker comprises theamino acid sequence DKTHT (SEQ ID NO:32). In certain embodiments, thelinker comprises the amino acid sequence GGGDKTHT (SEQ ID NO:41). Incertain embodiments, the linker is at least 11 amino acids long andcomprises the amino acid sequence EPKSCDKTHT (SEQ ID NO:33). In certainother embodiments, the linker comprises the amino acid sequenceVEPKSCDKTHT (SEQ ID NO:34), KVEPKSCDKTHT (SEQ ID NO:35), KKVEPKSCDKTHT(SEQ ID NO:36), DKKVEPKSCDKTHT (SEQ ID NO:37), VDKKVEPKSCDKTHT (SEQ IDNO:38), or KVDKKVEPKSCDKTHT (SEQ ID NO:39). In certain particularembodiments, the linker comprises the amino acid sequence EPKSSDKTHT(SEQ ID NO:40). In certain embodiments, the linker comprises the aminoacid sequence VEPKSSDKTHT (SEQ ID NO:67), KVEPKSSDKTHT (SEQ ID NO:68),KKVEPKSSDKTHT (SEQ ID NO:66), DKKVEPKSSDKTHT (SEQ ID NO:64),VDKKVEPKSSDKTHT (SEQ ID NO:69), or KVDKKVEPKSSDKTHT (SEQ ID NO:65). Incertain particular embodiments, the linker does not comprise the aminoacid sequence of GGS (SEQ ID NO: 45), GGGS (SEQ ID NO:46) or GGGGS (SEQID NO:47). In separate embodiments, the IL-22 IgG1 Fc fusion proteincomprises a linker sequence of GGGSTHT (SEQ ID NO:63). In otherparticular embodiments, the IL-22 Fc fusion protein comprises the aminoacid sequence of SEQ ID NO:12 or SEQ ID NO:14. In certain otherparticular embodiments, the IL-22 Fc fusion protein comprises the aminoacid sequence of SEQ ID NO:12.

In certain embodiments, the IL-22 Fc fusion protein comprises the CH2and CH3 domain of IgG4. In certain other embodiments, the linkercomprises the amino acid sequence SKYGPP (SEQ ID NO:43). In certainparticular embodiments, the linker comprises the amino acid sequenceRVESKYGPP (SEQ ID NO:44). In certain embodiments, none of the linkerscomprise the amino acid sequence GGS (SEQ ID NO:45), GGGS (SEQ ID NO:46)or GGGGS (SEQ ID NO:47). In other particular embodiments, the IL-22 Fcfusion protein comprises the amino acid sequence of SEQ ID NO:8 or SE IDNO:10. In particular embodiments, the IL-22 Fc fusion protein comprisesthe amino acid sequence of SEQ ID NO:8. In another embodiment, the IL-22Fc fusion protein is produced by the method comprising the step ofculturing a host cell capable of expressing the IL-22 Fc fusion proteinunder conditions suitable for expression of the IL-22 Fc fusion protein.In certain embodiments, the IL-22 Fc fusion protein is produced by themethod that further comprises the step of obtaining the IL-22 Fc fusionprotein from the cell culture or culture medium. In certain embodiments,the host cell is a Chinese hamster ovary (CHO) cell. In certain otherembodiments, the host cell is an E. coli cell.

In yet another aspect, the invention provides a composition comprisingan IL-22 Fc fusion protein, said IL-22 Fc fusion protein comprising anIL-22 polypeptide linked to an Fc region by a linker, wherein the Fcregion comprises a hinge region, an IgG CH2 domain and an IgG CH3domain, and wherein the composition has an afucosylation level in theCH2 domain of no more than 5%. In certain embodiments, the afucosylationlevel is no more than 2%, more preferably less than 1%. In certainembodiments, the afucosylation level is measured by mass spectrometry.In certain embodiments, the Fc region comprises the CH2 and CH3 domainof IgG4. In certain embodiments, the Fc region comprises a CH2 and CH3domain of IgG1. In certain other embodiments, the hinge region comprisesthe amino acid sequence of CPPCP (SEQ ID NO:31). In certain embodiments,the IL-22 Fc fusion protein comprises the amino acid sequence of SEQ IDNO:24 or SEQ ID NO:26. In certain embodiments, the IL-22 Fc fusionprotein comprises the amino acid sequence of SEQ ID NO:24. In certainembodiments, the composition is produced by the process comprising thesteps of culturing a host cell capable of expressing the IL-22 Fc fusionprotein under conditions suitable for expression of the IL-22 Fc fusionprotein, and obtaining the IL-22 Fc fusion protein from the cell cultureor culture medium, wherein the composition has an afucosylation level inthe CH2 domain of the Fc region of no more than 5%. In certainembodiments, the afucosylation level is no more than 2%, more preferablyless than 1%. In certain embodiments, the IL-22 Fc fusion protein isobtained by purification, preferably purifying fucosylated species awayfrom afucosylated species. In certain embodiments, the IL-22 Fc fusionprotein is purified by affinity chromatography. In certain embodiments,the host cell is a CHO cell.

In a further aspect, the invention provides an IL-22 Fc fusion protein,or a composition comprising IL-22 Fc fusion proteins, said IL-22 Fcfusion protein is produced by the process comprising the step ofculturing a host cell capable of expressing the IL-22 Fc fusion proteinunder conditions suitable for expression of the IL-22 Fc fusion protein.In certain embodiments, the process further comprises the step ofobtaining the IL-22 Fc fusion protein from the cell culture or culturemedium. In certain embodiments, the host cell is a CHO cell; while inother embodiments, the host cell is an E. coli cell.

In a further aspect, the invention provides a composition comprising anIL-22 Fc fusion protein described herein. In yet another aspect, theinvention provides a pharmaceutical composition comprising an IL-22 Fcfusion protein described herein, and at least one pharmaceuticallyacceptable carrier. In certain embodiments, the composition orpharmaceutical composition comprises an IL-22 Fc fusion proteincomprising an amino acid sequence of SEQ ID NO:8, SEQ ID NO:10, SEQ IDNO:12, SEQ ID NO:14, SEQ ID NO:24 or SEQ ID NO:26. In certain particularembodiments, the composition or pharmaceutical composition comprises anIL-22 Fc fusion protein comprising the amino acid sequence of SEQ IDNO:8. In certain particular embodiments, the IL-22 Fc fusion protein isproduced by E. coli. In certain other embodiments, the Fc region of theIL-22 Fc fusion protein is not glycosylated. In certain furtherembodiments, the IL-22 Fc fusion protein does not induce antibodydependent cellular cytotoxicity (ADCC). In certain embodiments, thepharmaceutical composition further comprises a suboptimal amount of atherapeutic agent such as dexamethasone. In certain embodiments, theIL-22 polypeptide comprises the amino acid sequence of SEQ ID NO:4.

Further, according to each and every aspect of the invention, in certainembodiments, the IL-22 Fc fusion protein can be a dimeric IL-22 Fcfusion protein (with respect to IL-22); while in other embodiments, theIL-22 Fc fusion protein can be a monomeric Fc fusion protein (withrespect to IL22).

In a further aspect, the invention provides a monomeric IL-22 Fc fusionprotein. In certain particular embodiments, the monomeric fusion proteincomprises an IL-22 Fc fusion arm and an Fc arm. In certain embodiments,the IL-22 Fc fusion arm and the Fc arm comprises either a knob or a holein the Fc region. In certain embodiments, the Fc region of the IL-22 Fcfusion arm (the monomer IL-22 Fc fusion) comprises a knob and the Fcregion of the Fc arm (the monomer Fc without linking to IL-22) comprisesa hole. In certain embodiments, the Fc region of the IL-22 Fc fusion arm(the monomer IL-22 Fc fusion) comprises a hole and the Fc region of theFc arm (the monomer Fc without linking to IL-22) comprises a knob. Incertain other embodiments, the monomeric IL-22 Fc fusion proteincomprises the amino acid sequence of SEQ ID NO:61 and SEQ ID NO:62. Incertain other embodiments, the Fc region of both arms further comprisesan N297G mutation. In certain embodiments, the monomeric IL-22 Fc isproduced by the process comprising the step of culturing one or morehost cells comprising one or more nucleic acid molecules capable ofexpressing the first polypeptide comprising the amino acid sequence ofSEQ ID NO:61 and the second polypeptide comprising the amino acidsequence of SEQ ID NO:62. In certain other embodiments, the methodfurther comprises the step of obtaining the monomeric IL-22 Fc fusionprotein from the cell culture or culture medium. In certain embodiments,the host cell is an E. coli cell. In a related aspect, the inventionprovides a composition or pharmaceutical composition comprising themonomeric IL-22 Fc fusion protein.

In yet another aspect, the invention provides an isolated nucleic acidencoding the IL-22 Fc fusion protein described herein. In certainembodiments, the nucleic acid encodes the IL-22 Fc fusion proteincomprising the amino acid sequence of SEQ ID NO:8, SEQ ID NO:10, SEQ IDNO:12, SEQ ID NO:14, SEQ ID NO:24 or SEQ ID NO:26, preferably SEQ IDNO:8 or SEQ ID NO:12, more preferably SEQ ID NO:8. In certain otherembodiments, the nucleic acid comprises the polynucleotide sequence ofSEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:23 orSEQ ID NO:25. In certain particular embodiments, the nucleic acidcomprises the polynucleotide sequence of SEQ ID NO:7 or SEQ ID NO:11,preferably SEQ ID NO:7. In certain embodiments, the isolated nucleicacid comprises a polynucleotide sequence that is at least 80%, 85%, 86%,87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%sequence identity to the polynucleotide sequence of SEQ ID NO:7, SEQ IDNO:9, SEQ ID NO:11, SEQ ID NO:13; SEQ ID NO:23 or SEQ ID NO:25. Incertain embodiments, the isolated nucleic acid comprises apolynucleotide sequence that is at least 80%, 85%, 86%, 87%, 88%, 89%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequenceidentity to the polynucleotide sequence of SEQ ID NO:7, SEQ ID NO:9, SEQID NO:11, SEQ ID NO:13; SEQ ID NO:23 or SEQ ID NO:25, wherein theisolated nucleic acid is capable of encoding an IL-22 Fc fusion proteinthat is capable of binding to IL-22R and/or triggering IL-22R activityand wherein the Fc region of the IL-22 Fc fusion protein is notglycosylated. In certain embodiments, the isolated nucleic acidcomprises a polynucleotide sequence that is at least 80%, 85%, 86%, 87%,88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%sequence identity to the polynucleotide sequence of SEQ ID NO:7, SEQ IDNO:9, SEQ ID NO:11, SEQ ID NO:13; SEQ ID NO:23 or SEQ ID NO:25, whereinthe isolated nucleic acid is capable of encoding an IL-22 Fc fusionprotein comprising the amino acid sequence of SEQ ID NO:8, 10, 12, or14. In related aspects, the invention provides vectors comprising thenucleic acid described above, and a host cell comprising the vector. Incertain embodiments, the host cell is a prokaryotic cell or eukaryoticcell. In certain particular embodiments, the host cell is a prokaryoticcell, including without limitation, an E. coli cell. In certain otherembodiments, the host cell is a eukaryotic cell, including withoutlimitation, a CHO cell. In certain embodiments, the host cell comprisesa vector comprising a nucleic acid encoding the IL-22 Fc fusion proteincomprising the amino acid sequence of SEQ ID NO:8.

In a further related aspect, the invention provides methods of makingthe IL-22 Fc fusion protein comprising the step of culturing the hostcell under conditions suitable for expression of the IL-22 Fc fusionprotein. In certain embodiments, the method further comprises the stepof obtaining the IL-22 Fc fusion protein from the cell culture orculture medium. The IL-22 Fc fusion protein can be obtained from thecell culture or culture medium by any methods of protein isolation orpurification known in the art, including without limitation, collectingculture medium, freezing/thawing, centrifugation, cell lysis,homogenization, ammonium sulfate precipitation, HPLC, and affinity, gelfiltration, and ion exchanger column chromatography. In certainembodiments, the method further comprises the step of removingafucosylated IL-22 Fc fusion protein. In certain other embodiments, theafucosylated IL-22 Fc fusion protein is removed by affinity columnchromatography. In certain embodiments, the host cell is an E. colicell. In other embodiments, the host cell is a CHO cell.

In yet another aspect, the invention provides a composition orpharmaceutical composition comprising an IL-22 Fc fusion protein of theinvention and at least one pharmaceutically acceptable carrier. Incertain embodiments, the IL-22 Fc fusion protein comprises the aminoacid sequence of SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14,SEQ ID NO:24, or SEQ ID NO:26. In other embodiments, the Fc region ofthe IL-22 Fc fusion protein is not glycosylated. In certain embodiments,the Fc region of the IL-22 Fc fusion protein is not glycosylated whilethe IL-22 polypeptide is glycosylated. In certain such embodiments, theIL-22 Fc fusion protein is produced in CHO cells. In certainembodiments, the IL-22 Fc fusion protein does not induce antibodydependent cellular cytotoxicity. In yet other embodiments, thepharmaceutical composition further comprises dexamethasone or a TNFantagonist. In certain particular embodiments, the dexamethasone or aTNF antagonist is present at a suboptimal amount.

In certain other embodiments, the pharmaceutical composition comprisingIL-22 Fc fusion proteins has an afucosylation level in the CH2 domain ofno more than 5%, preferably no more than 2%, more preferably less than1%. In certain particular embodiments, the IL-22 Fc fusion proteincomprises the amino acid sequence of SEQ ID NO:24 or SEQ ID NO:26,preferably SEQ ID NO:24. In certain other embodiments, the IL-22 Fcfusion protein is produced in CHO cells. In certain particularembodiments, the subject is a human. In certain embodiments, thepharmaceutical composition is administered systematically or topically.In certain other embodiments, the pharmaceutical composition isadministered intravenously, subcutaneously, intraperitoneally ortopically.

In a further aspect, the invention provides a pharmaceutical compositioncomprising an IL-22 polypeptide or IL-22 Fc fusion protein describedherein and at least one pharmaceutically acceptable carrier. In certainembodiments, the pharmaceutically acceptable carrier is a gelling agent.In certain embodiments, the gelling agent is a polysaccharide. In someembodiments, the gelling agent is, without limitation, methylcellulose,hydroxyethyl cellulose, carboxymethyl cellulose, hydroxypropylcellulose, POE-POP block polymers, alginate, hyaluronic acid,polyacrylic acid, hydroxyethyl methylcellulose or hydroxypropylmethylcellulose. In some embodiments, the polysaccharide is a cellulosicagent such as, without limitation, hydroxyethyl methylcellulose orhydroxypropyl methylcellulose. In certain embodiments, the gelling agentis hydroxypropyl methylcellulose. In some embodiments, thepharmaceutical composition is for topical administration. In certainembodiments, the pharmaceutical composition for topical administrationcomprises an IL-22 polypeptide. In some embodiments, the pharmaceuticalcomposition for topical administration comprises an IL-22 Fc fusionprotein. In certain embodiments, the pharmaceutical composition fortopical administration comprises an IL-22 polypeptide without an Fcfusion.

In another aspect, the invention provides methods of treating IBD in asubject in need thereof comprising administering to the subject thepharmaceutical composition comprising an IL-22 Fc fusion protein of theinvention. In certain embodiments, the IBD is ulcerative colitis. Incertain other embodiments, the IBD is Crohn's disease. In certainparticular embodiments, the Fc region of the IL-22 Fc fusion protein isnot glycosylated. In certain embodiments, the N297 residue and/or theT299 residue of the Fc region is changed. In certain embodiments, theN297 residue of the Fc region is changed. In certain other embodiments,the N297 residue is changed to Gly or Ala, preferably Gly. In certainother embodiments, the T299 residue is changed, preferably to Val, Glyor Ala. In certain particular embodiments, the IL-22 Fc fusion proteincomprises the amino acid sequence of SEQ ID NO:8, SEQ ID NO:10, SEQ IDNO:12 or SEQ ID NO:14, preferably SEQ ID NO:8. In certain embodiments,the IL-22 Fc fusion protein is produced in E. coli or a CHO cell. Incertain embodiments, the subject is a human. In certain otherembodiments, the pharmaceutical composition is administeredintravenously, subcutaneously, intraperitoneally or topically.

In another aspect, the invention provides methods of treating any one orcombination of the following diseases using an IL-22 polypeptide or anIL-22 Fc fusion protein of this invention: Type II diabetes, Type IIdiabetes with morbid obesity, wounds (including diabetic wounds anddiabetic ulcers), burns, ulcers (including pressure ulcer and venousulcer), graft versus host disease (GVHD), atherosclerosis,cardiovascular disease, metabolic syndrome, endotoxemia (acute andmild), sepsis, acute coronary heart disease, hypertension, dyslipemia,obesity, hyperglycemia, lipid metabolism disorders, hepatitis, acutehepatitis, renal failure, acute renal failure, acute kidney injury,renal draft failure, post cadaveric renal transplant delayed graftfunction, contrast induced nephropathy, pancreatitis, acutepancreatitis, liver fibrosis and lung fibrosis. In certain embodiments,acute pancreatitis can be mild to moderate to severe disease. In certainembodiments, acute pancreatitis includes disease post ERCP (endoscopicretrograde cholangiopancreatography). In some further embodiments, thepatient to be treated for the above disease is in need of a change inhis HDL/LDL lipid profile, which IL-22 polypeptide or IL-22 Fc fusionproteins can alter in the patient to increase HDL and decrease LDL. In arelated aspect, the invention provides uses of an IL-22 polypeptide oran IL-22 Fc fusion protein in the preparation of a medicament for thetreatment of any one or combinations of the above diseases.

In a further aspect, the invention provides methods of inhibitingmicrobial infection in the intestine, or preserving goblet cells in theintestine during a microbial infection, of a subject in need thereofcomprising the step of administering to the subject the pharmaceuticalcomposition comprising the IL-22 Fc fusion protein of the invention. Inother related aspects, the invention provides methods of enhancingepithelial cell integrity, mucosal healing, epithelial cellproliferation, epithelial cell differentiation, epithelial cellmigration or epithelial wound healing in the intestine in a subject inneed thereof comprising administering to the subject the pharmaceuticalcomposition comprising the IL-22 Fc fusion protein of the invention. Incertain embodiments, the epithelial cell is intestinal epithelial cell.

In another aspect, a method for preventing or treating a cardiovascularcondition, which condition includes a pathology of atheroscleroticplaque formation, is provided. The method includes administering to asubject in need thereof a therapeutically effective amount of an IL-22polypeptide or an IL-22 Fc fusion protein. The cardiovascular conditionincludes, for example, coronary artery disease, coronary microvasculardisease, stroke, carotid artery disease, peripheral arterial disease,and chronic kidney disease. The method can include further slowing downthe progression of atherosclerotic plaque formation. The method canfurther include administering one or more additional therapeutic agentto the subject for the prevention or treatment of the cardiovascularcondition.

In another aspect, a method for treating metabolic syndrome is provided.The method includes administering to a subject in need thereof atherapeutically effective amount of an IL-22 polypeptide or an IL-22 Fcfusion protein. The method can further include reducing one or more riskfactors associated with metabolic syndrome, including one or more ofabdominal obesity, hyperglycemia, dyslipidemia, and hypertension. Themethod can further include reducing the level of bacteriallipopolysaccharide (LPS) in the subject. The method can further includeadministering one or more additional agent to the subject for theprevention or treatment of metabolic syndrome.

In another aspect, a method for delaying or slowing down the progressionof atherosclerosis is provided. The method includes administering to asubject in need thereof a therapeutically effective amount of an IL-22polypeptide or an IL-22 Fc fusion protein. The method can furtherinclude administering one or more additional agent to the subject fordelaying or slowing down the progression of atherosclerosis.

In another aspect, a method of preventing indicia of atherosclerosis isprovided. The method includes administering a therapeutically effectiveamount of an IL-22 polypeptide or an IL-22 Fc fusion protein to asubject at risk of atherosclerosis, wherein the IL-22 polypeptide ofIL-22 Fc fusion protein is effective against the development of indiciaof atherosclerosis. In certain embodiments, the subject has beenidentified to be at risk to develop a cardiovascular condition. Incertain embodiments, the subject is genetically at risk of developing acardiovascular condition. In one or more embodiments, the indicia ofatherosclerosis include plaque accumulation. In some embodiments, theindicia of atherosclerosis include vascular inflammation. The method canfurther include administering one or more additional agent to thesubject for preventing indicia of atherosclerosis.

In yet another aspect, a method of treating one or more of acuteendotoxemia and sepsis is provided. The method includes administering toa subject in need thereof a therapeutically effective amount of an IL-22polypeptide or an IL-22 Fc fusion protein. The method can furtherinclude administering one or more additional agent to the subject fortreating one or more of acute endotoxemia and sepsis.

In one other aspect, a method is provided for accelerating or improvingwound healing, or both, in a subject. The method includes administeringto a subject in need thereof a therapeutically effective amount of anIL-22 polypeptide, an IL-22 Fc fusion protein or an IL-22 agonist. Incertain embodiments, the wound is a chronic wound. In certain otherembodiments, the wound is an infected wound. In certain embodiments, thesubject is diabetic, including a subject with type II diabetes. In oneor more embodiments, the wound is a diabetic foot ulcer. In certainembodiments, the therapeutically effective amount of an IL-22polypeptide, IL-22 Fc fusion protein or IL-22 agonist is administereduntil there is complete wound closure. In some embodiments, theadministration is systemic; and in other embodiments, the administrationis topical. In certain embodiments, the IL-22 polypeptide, IL-22 Fcfusion protein or IL-22 agonist is in a formulation for topicaladministration. In certain embodiments, the topical formulationcomprises an IL-22 polypeptide without an Fc fusion. In certainembodiments, the IL22 agonist is selected from the group consisting ofan IL-22 polypeptide, an IL-22 Fc fusion protein, an IL-22 agonist, anIL-19 polypeptide, an IL-19 Fc fusion protein, an IL-19 agonist, anIL-20 polypeptide, an IL-20 Fc fusion protein, an IL-20 agonist, anIL-24 polypeptide, an IL-24 Fc fusion protein, an IL-24 agonist, anIL-26 polypeptide, an IL-26 Fc fusion protein, an IL-26 agonist, and anIL-22R1 agonist. In certain other embodiments, the IL-22 agonist isselected from the group consisting of an IL-22 polypeptide, an IL-22 Fcfusion protein, an IL-22 agonist, an IL-20 polypeptide, an IL-20 Fcfusion protein, an IL-20 agonist, an IL-24 polypeptide, an IL-24 Fcfusion protein, an IL-24 agonist and an IL-22R1 agonist. In certainembodiments, the IL-22R1 agonist is an anti-IL22R1 agonistic antibody.

In a further aspect, the invention provides methods of treating ametabolic syndrome comprising the step of administering to a subject inneed thereof a therapeutically effective amount of one or more IL-22agonists. In certain embodiments, the IL22 agonist is selected from thegroup consisting of an IL-22 polypeptide, an IL-22 Fc fusion protein, anIL-22 agonist, an IL-19 polypeptide, an IL-19 Fc fusion protein, anIL-19 agonist, an IL-20 polypeptide, an IL-20 Fc fusion protein, anIL-20 agonist, an IL-24 polypeptide, an IL-24 Fc fusion protein, anIL-24 agonist, an IL-26 polypeptide, an IL-26 Fc fusion protein, anIL-26 agonist, and an IL-22R1 agonist. In certain other embodiments, theIL-22 agonist is selected from the group consisting of an IL-22polypeptide, an IL-22 Fc fusion protein, an IL-22 agonist, an IL-20polypeptide, an IL-20 Fc fusion protein, an IL-20 agonist, an IL-24polypeptide, an IL-24 Fc fusion protein, an IL-24 agonist and an IL-22R1agonist. In certain embodiments, the IL-22R1 agonist is an anti-IL22R1agonistic antibody. In certain other embodiments, the metabolic syndromeis diabetes. In certain particular embodiments, the metabolic syndromeis type II diabetes.

According to another embodiment, the subject is administered an IL-22 Fcfusion protein of the invention. In certain embodiments, the subject isa human. In certain embodiments, the IL-22 polypeptide or IL22 Fc fusionprotein is administered intravenously, subcutaneously,intraperitoneally, systemically or topically.

In certain embodiments of these aspects, the Fc region of the IL-22 Fcfusion protein is not glycosylated. In certain embodiments, the N297residue and/or the T299 residue of the Fc region is changed. In certainembodiments, the N297 residue of the Fc region is changed. In certainother embodiments, the N297 residue is changed to Gly or Ala, preferablyGly. In certain other embodiments, the T299 residue is changed,preferably to Val, Gly or Ala. In certain particular embodiments, theIL-22 Fc fusion protein comprises the amino acid sequence of SEQ IDNO:8, SEQ ID NO:10, SEQ ID NO:12 or SEQ ID NO:14, preferably SEQ IDNO:8. In certain embodiments, the IL-22 Fc fusion protein is produced inE. coli. In certain embodiments, the subject is a human. In certainother embodiments, the pharmaceutical composition is administeredintravenously, subcutaneously or topically.

In certain other embodiments, the pharmaceutical composition comprisingIL-22 Fc fusion proteins has an afucosylation level in the CH2 domain ofno more than 5%, preferably no more than 2%, more preferably less than1%. In certain particular embodiments, the IL-22 Fc fusion proteincomprises the amino acid sequence of SEQ ID NO:24 or SEQ ID NO:26,preferably SEQ ID NO:24. In certain other embodiments, the IL-22 Fcfusion protein is produced in CHO cells. In certain particularembodiments, the subject is a human. In certain other embodiments, thepharmaceutical composition is administered intravenously, subcutaneouslyor topically.

In yet other embodiments of the above aspects, the N-glycan attached tothe Fc region of the IL-22 Fc fusion protein is enzymatically removed bya glycolytic enzyme. In certain embodiments, the glycolytic enzyme ispeptide-N-glycosidase (PNGase). In certain particular embodiments, thesubject is a human.

In yet a further aspect, the invention also provides uses of an IL-22 Fcfusion protein described herein in the preparation of a medicament forthe treatment of IBD, including UC and CD, in a subject in need thereof.In a related aspect, the invention provides uses of an IL-22 Fc fusionprotein described herein in the preparation of a medicament forinhibiting microbial infection in the intestine, or preserving gobletcells in the intestine during a microbial infection in a subject in needthereof. In yet another aspect, the invention provides uses of an IL-22Fc fusion protein described herein in the preparation of a medicamentfor enhancing epithelial cell integrity, epithelial cell proliferation,epithelial cell differentiation, epithelial cell migration or epithelialwound healing in the intestine, in a subject in need thereof. In otherrelated aspects, the invention provides uses of an IL-22 polypeptide orIL-22 Fc fusion protein in the preparation of a medicament for treatinga cardiovascular condition, metabolic syndrome, atherosclerosis, acutekidney injury, acute pancreatitis, accelerating, promoting or improvingwound healing, including without limitation, healing of a chronic wound,diabetic wound, infected wound, pressure ulcer or diabetic foot ulcer,in a subject in need thereof.

Each and every embodiment can be combined unless the context clearlysuggests otherwise. Each and every embodiment can be applied to each andevery aspect of the invention unless the context clearly suggestsotherwise.

Specific embodiments of the present invention will become evident fromthe following more detailed description of certain preferred embodimentsand the claims.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows amino acid sequence alignment of mature IL-22 fromdifferent mammalian species: human (GenBank Accession No. Q9GZX6, SEQ IDNO:4, chimpanzee (GenBank Accession No. XP_003313906, SEQ ID NO:48),orangutan (GenBank Accession No. XP_002823544, SEQ ID NO:49), mouse(GenBank Accession No. Q9JJY9, SEQ ID NO:50) and dog (GenBank AccessionNo. XP_538274, SEQ ID NO:51).

FIGS. 2A-2G show mass spectrometry results of the glycosylation statusof the Fc region of a typical human monoclonal IgG1 Fc (FIG. 2A), IL-22IgG1 Fc fusion containing the linker sequence EPKSCDKTHT (SEQ ID NO:33,FIG. 2B), EPKSSDKTHT (SEQ ID NO:40, FIG. 2C), and GGGDKTHT (SEQ IDNO:41, FIG. 2D), and IL-22 IgG4 Fc fusion containing the linker sequenceRVESKYGPP without or with the N297G mutation (SEQ ID NO:44, FIGS. 2E and2F, respectively) and IL-22 IgG1 Fc fusion containing the linkersequence EPKSSDKTHT (SEQ ID NO:40) with the N297G mutation (FIG. 2G).

FIG. 3 shows sequence alignment of human IL-22 IgG4 Fc fusion (N297G,full length Fc sequence with the C-terminal Lys, SEQ ID NO:16, withoutLys SEQ ID NO:8), IL-22 IgG1 Fc fusion (N297G, full length Fc sequencewith the C-terminal Lys, SEQ ID NO:20, without Lys SEQ ID NO:12) andIL-22 (SEQ ID NO:4). The IL-22 sequence shown is the matured formwithout the leader sequence. The hinge sequence CPPCP (SEQ ID NO:31) isshown in the box, followed by the CH2 and CH3 domains. The N297Gsubstitution and the optional C-terminus Lys residue are marked.

FIG. 4 presents a graph showing the results of STATS luciferase assay.Luciferase activity stimulated by IL-22 IgG4 Fc fusion or IL-22 IgG1 Fcfusion was measured in 293 cells expressing human IL-22R. The resultsshow that IL-22 IgG4 and IL-22 IgG1 Fc fusion exhibited similar in vitroactivity.

FIGS. 5A-5C show the therapeutic effects of mouse IL-22 Fc fusionprotein in the dextran sodium sulfate (DSS)-induced mouse IBD model.Mouse IL-22 Fc fusion protein improved colon histology in theDSS-induced IBD mice (FIG. 5B) and the improvement was translated toreduced colon histology score (FIG. 5C). IL-22 Fc fusion proteintreatment resulted in reduced weight loss of the mice during treatmentas compared to dexamethasone, currently the best standard of care inthis model (FIG. 5A).

FIG. 6 shows the rate of serum clearance of human IL-22 IgG4 and IgG1 Fcfusion proteins in cynomolgus monkeys dosed at 0.15 mg/kg and 1.5 mg/kgon day 0 and day 7.

FIGS. 7A-7C show the serum levels of three IL-22R downstream genes incynomolgus monkeys after dosing at 0.15 mg/kg and 1.5 mg/kg at day 1 andday 8 (same dosing regimen as day 0 and day 7 in FIG. 6. FIG. 7A showsdose-dependent increases in serum amyloid A (SAA), FIG. 7B showsdoes-dependent increases in lipopolysaccharide binding protein (LPS-BP),FIG. 7C shows dose-dependent increases in RegIII/Pancreatitis AssociatedProtein (PAP or PancrePAP), following hIL-22 Fc administration.

FIG. 8 shows a high resolution MicroCT demonstrating the atheroscleroticplaque burden in the aorta arch and brachiocephalic artery of an 8 monthold Ldlr−/−Apobec1−/− mouse on high fat diet.

FIGS. 9A and 9B show that Ldlr−/−Apobec1−/− mice were sensitive todietary challenges and showed a substantially increased level ofatherosclerosis as measured from microCT (FIG. 9A), but with onlymodestly increased serum LDL levels (FIG. 9B).

FIGS. 10A-10C show the response of Ldlr−/−Apobec1−/− mice to an acutelow grade inflammation stimulus, demonstrating an increase in sera MCP-1(FIG. 10A) and IL-6 (FIG. 10B) greater than observations in wt C57 miceand accompanied by loss of vascular function as assessed by flowmediated dilation and infusion of nitroglycerine (FIG. 10C).

FIGS. 11A-11C show that chronic endotoxin exposure results indyslipidemia (FIG. 11A) and greater plaque burden (FIG. 11B) andinstability (FIG. 11C).

FIGS. 12A-12C show fasting blood glucose was reduced in the IL-22-Fctreated group compared to controls (FIG. 12A) and glucose clearance wasimproved with IL-22-Fc treatment as seen from the glucose tolerance test(FIGS. 12B and 12C).

FIGS. 13A and 13B show that a reduction in total cholesterol occursafter treatment with IL-22-Fc. In Ldlr−/−Apobec1−/− mice totalcholesterol was elevated, in both the fasting and fed conditions, andwas reduced in the IL-22-Fc group compared with the controls as measuredat the end of the treatment period (FIG. 13A). Plasma triglycerideslevels were also reduced upon IL-22-Fc treatment with a marked reductionin the fed state (FIG. 13B).

FIGS. 14A-14G show that the hyperlipidemia seen in the Ldlr−/−Apobec1−/−mouse was reduced following IL-22-Fc treatment. LDL was reduced in boththe fasting and fed state (FIG. 14A), HDL was raised (FIG. 14B), andLDL/HDL ratio were reduced in both fast and fed (FIG. 14C). vLDL wasreduced under fed conditions (FIG. 14D). Results of HDL (FIG. 14E), LDL(FIG. 14F) and LDL/HDL ratio (FIG. 14G) were depicted after 5 days withmice given two doses.

FIG. 15 shows that plasma LPS levels were reduced after IL-22-Fctreatment.

FIG. 16 shows improved endothelial function measure by vascularreactivity after IL-22-Fc treatment.

FIGS. 17A-17C depict the quantitative analysis of plaque burdenperformed using contrast-enhanced microCT on postmortem samples of thedissected aortic arch, ascending and descending aorta (FIG. 17A), thebrachiocephalic artery (FIG. 17B) and aortic valve (FIG. 17C).

FIGS. 18A and 18B show body weights (FIG. 18A) and food intake (FIG.18B) following IL-22-Fc treatment.

FIG. 19 depicts a schematic of diabetic mouse model treatment regimen.

FIGS. 20A-20C show body weight (FIG. 20B) and serum glucose levels(FIGS. 20A and 20C) in db/db mice demonstrating that IL-22-Fcsignificantly reduced glucose in the obese mice.

FIG. 21 shows IL-22Fc treatment improves glucose tolerance and insulinsensitivity based on the Glucose Tolerance Test (GTT). p<0.05

FIGS. 22A and 22B show that IL-22Fc treatment improved insulinsensitivity based on the Insulin Tolerance Test (ITT) as measuredthrough mg/dL glucose levels (FIG. 22A) and % glucose reduction (FIG.22B).

FIGS. 23A-23F show that IL-22Fc increased insulin expression in islets.(FIG. 23A) Green shows glucagon, red shows insulin. The circled areasurrounded by red line shows islet area. Bar, 50 μm. (FIG. 23B) Averageinsulin staining intensity. (FIG. 23C) Average glucagon stainingintensity. (FIG. 23D) Fed insulin levels in HFD-fed mice. (FIG. 23E)Fasted insulin levels in HFD-fed mice. (FIG. 23F) IL-22 Fc reversedinsulin insensitivity in HFD-fed mice. **P<0.01, ***P<0.001. Error bars,s.e.m.

FIGS. 24A and 24B depict quantitative analysis of insulin-signalintensity in IL-22-Fc treated animals.

FIGS. 25A and 25B show that the insulin-positive area was increased inIL-22-Fc treated animals compared to control.

FIGS. 26A and 26B show histological sections demonstrating a decrease inhepatic periportal steatosis with IL-22-Fc treatment (FIG. 26B) ascompared to control (FIG. 26A).

FIGS. 27A and 27B show an assessment of IL-22R in HFD induced glucosetolerance. (FIG. 27A) glucose levels (mg/dL) over time post glucose ipinjection. (FIG. 27B) Calculation of the total area under the curve(AUC).

FIG. 28 shows mass of IL-22 receptor KO mice compared to littermatecontrol.

FIGS. 29A-29D Ldlr −/−, Apobec1 −/− (dko) mice were treated with 50 ugIL-22Fc or 50 ug anti-ragweed (n=6 per group) for 48 hours. Serum LPSwas reduced by 50% (p=0.0052) and serum LDL/HDL was reduced by 30%(p=0.049) in IL-22Fc treated mice.

FIG. 30 shows a nucleotide sequence of a cDNA encoding a native humanIL-22 (SEQ ID NO:70).

FIG. 31 shows the amino acid sequence derived from the coding sequenceshown in FIG. 30 (SEQ ID NO:71).

FIG. 32A shows the amino acid sequence of a mouse IL-22-mouse-IgG2afusion protein (SEQ ID NO:73).

FIG. 32B shows the nucleotide sequence encoding mouse IL-22-mouse IgG2afusion protein (SEQ ID NO:72).

FIG. 33 shows that lack of signaling through IL-22R results in delayedwound healing. IL-22R KO mice wounds were significantly delayed(p=0.0018 on day 10 & p=0.005 on day 12) in healing compared to WTlittermate control mice.

FIGS. 34A-34C represent individual mice (n=10) wound gap at days 10, 12and 15.

FIG. 34D shows representative photo images of the wounds for both IL-22RKO mice and WT at day 14.

FIGS. 35A and 35B illustrate a wound healing comparison between ControlWT mice (BKS) and Diabetic db/db mice. FIG. 35A shows that wound healingin the db/db mice was considerably delayed throughout the period ofstudy and did not heal fully even at day 28. FIG. 35B is a bar graphshowing the level of IL-22 expression as fold change in wild type ordb/db mice days after wound excision.

FIG. 36 is a schematic representation of the study design for testingIL-22-Fc in db/db mice in a total of 3 groups (n=7). Anti-ragweed wasused for control Fc protein and anti-FGFR1 antibody was used as positivecontrol for glucose regulation.

FIG. 37 shows IL-22 Fc normalized fed glucose level of treated mice ascompared to controls from days 4 until day 27. Glucose levels wererecorded using an Onetouch® glucometer.

FIG. 38 shows graphically comparative wound gap measurement of IL-22-Fccompared to 2 control antibodies: anti-ragweed and anti-FGFR1. Each datapoint represents an average of 7 mice/group.

FIGS. 39A-39D show individual wound gap measurements at days 15, 19, 21,and day 27.

FIG. 39E shows photographs of representative mice at day 27.

FIG. 40 is a schematic representation of the study design for testingtopical vs. systemic dosing of IL-22-Fc compared to control antibodytreatment in db/db mice; Total 3 groups (n=7).

FIGS. 41A and 41B show graphically comparative wound gap measurement ofIL-22-Fc topical vs. systemic dosing with control Fc topical treatment.Anti-ragweed antibody was used as an Fc control antibody. Each datapoint represents an average of 7 mice/group.

FIGS. 42A and 42B show photographically surgically removed wound tissuefrom representative mice showing both top as well as back view on day 22from IL-22-Fc (FIG. 42B) and control antibody (FIG. 42A).

FIG. 43A shows the strategy for generation of IL-22R KO mice.

FIG. 43B shows RT-PCR results of IL-22Ra1 mRNA expression in colon fromIL-22R KO and WT mice. ***P<0.001. Error bars, s.e.m.

FIG. 43C shows RT-PCR results of Reg3b mRNA expression in colon fromIL-22R KO and WT mice 2 days after a single dose injection of IL-22 Fcor control IgG. ***P<0.001. Error bars, s.e.m.

FIGS. 44A-44F show results demonstrating that obese mice mounteddefective IL-22 responses. (FIGS. 44A-44D) Lymphocytes in draining lymphnodes of db/db (FIGS. 44A and 44B), DIO (FIGS. 44C and 44D) and controlmice immunized with OVA/CFA were analyzed for IL-22 expression on day 7by flow cytometry. Numbers on the FACS plots in (FIGS. 44A and 44C) arepercentage of IL-22⁺ cells within CD4⁺ T cells. (FIGS. 44E-44F) db/db,lean controls, HFD and chow diet-fed normal mice were injected withflagellin or PBS. Serum was harvested after 2 h. ELISA of IL-22 fromdb/db and lean controls (FIG. 44E), and HFD and chow diet-fed mice (FIG.44F). Data shown are representative of three (FIGS. 44A and 44B) or two(FIGS. 44C-44F) independent experiments. N=4 in all experiments. *P<0.05, **P<0.01, ***P<0.001, Error bars, s.e.m.

FIGS. 45A-45E show defects in IL-17 and IL-22 production in leptinsignal-deficient mice. (FIGS. 45A and 45B) IL-17A and IL-22 expressionwere analyzed on day 7 as percentage within CD4+ cells in db/db andob/ob mice immunized with OVA/CFA. (FIG. 45C) IL-22 ELISA from culturesupernatant of purified naïve WT CD4+ T cells that were stimulated underIL-22 producing conditions with or without recombinant mouse leptin (1μg/ml). (FIG. 45D) IL-22 ELISA from culture supernatant of Rag2 KOsplenocytes stimulated with IL-23 with or without recombinant mouseleptin (1 μg/ml). (FIG. 45E) ELISA of serum IL-22 from ob/ob or leancontrols 2 hours after flagellin stimulation. * P<0.05, **P<0.01,***P<0.001, Error bars, s.e.m.

FIGS. 46A-46J show results demonstrating that the susceptibility ofdb/db (ob/ob) mice to C. rodentium infection was associated withdefective IL-22 production and rescued by exogenous IL-22-Fc. (FIG. 46A)IL-22 mRNA expression in colons from WT, db/db and ob/ob mice (n=5)after C. rodentium infection. (FIG. 46B) Body weight and (FIG. 46C)survival of db/db and lean control mice (n=10) infected with C.rodentium. (FIGS. 46D and 46E) Colon histology of lean control (FIG.46D) and db/db (FIG. 46E) mice on day 10, showing epithelialhyperplasia, enterocyte shedding into the gut lumen, bacterial colonies(arrows) and submucosal edema (vertical bar). Horizontal bar, 200 μm.(FIG. 46F) Clinical score determined by colon histology (n=5). (FIGS.46G and 46H) Bacterial burden of db/db and lean control mice (n=5) inliver (FIG. 46G) and spleen (FIG. 46H) on day 10. (FIG. 46I) ELISA ofanti-C. rodentium IgG in lean control and db/db mice (n=5) on day 10.(FIG. 46J). Survival of lean control or db/db mice (n=10) treated withIL-22-Fc or control IgG after infection. Data shown are representativeof three independent experiments. * P<0.05, **P<0.01, ***P<0.001, Errorbars, s.e.m.

FIGS. 47A-47D show results demonstrating that diabetic disorders werereduced by IL-22-Fc treatment. HFD-fed mice were treated with IL-22-Fctwice per week (n=10). (FIG. 47A) Blood glucose on day 20 (fed) and day21 (16-hour fasting). (FIG. 47B) Body weight on day 30. (FIG. 47C)Glucose tolerance test on day 21. (FIG. 47D) Insulin tolerance test onday 28. Data shown are representative of two independent experiments. *P<0.05, **P<0.01, ***P<0.001, Error bars, s.e.m.

FIGS. 48A-48D show results demonstrating that IL-22 prevents thediabetic disorders of mice fed with HFD. (FIG. 48A) body weight, (FIG.48B) blood glucose, (FIG. 48C) glucose tolerance test on day 23, (FIG.48D) blood glucose on day 23 after 16 h fast, and (FIG. 48E) abdominalfat pad on day 25. * P<0.05, **P<0.01, ***P<0.001, Error bars, s.e.m.

FIGS. 49A-49I show results demonstrating that IL-22 regulates metabolicsyndrome through multiple mechanisms. (FIGS. 49A-49C) Two groups ofdb/db mice (n=8) were fed with food ad libitum and treated with controlIgG or IL-22-Fc twice per week. One group of db/db mice (n=8) was fedwith restricted food that matched the food intake of IL-22-Fc treatedgroup, and treated with control IgG. Accumulative food intake of firsteight days of ad lib fed mice is shown in FIG. 49A, blood glucose inFIG. 49B, and glucose tolerance test on day 25 in FIG. 49C. FIGS. 49Dand 49E show PYY levels in db/db (FIG. 49D) and HFD (FIG. 49E) micetreated with IL-22-Fc or control IgG on day 0 and day 2. Serum wascollected on day 2 before the 2nd treatment and on day 5, and analyzedfor PYY. FIG. 49F shows serum LPS of db/db mice treated with IL-22-Fc orcontrol IgG for 3 weeks. (FIGS. 49G-49I) IL-22R KO (n=9) and WT mice(n=6) were fed with HFD starting at 6 weeks of age. The results of bodyweight are shown in FIG. 49G, results of glucose tolerance test at 3months with HFD are shown in FIG. 49H, and results of Insulin tolerancetest at 4 months with HFD are shown in FIG. 49I. Data shown arerepresentative of two (FIGS. 49A-49C) or three (FIGS. 49D-49I)independent experiments. * P<0.05, **P<0.01, ***P<0.001, Error bars,s.e.m.

FIG. 50 shows results of pair-feeding restricted food intake. Threegroups of db/db mice were fed and treated as in FIG. 49A. Accumulativefood intake was measured.

FIGS. 51A-51J show results demonstrating IL-22 improved liver functionand reduced fat pad. (FIG. 51A) db/db mice treated with IL-22 Fc orcontrol IgG as in FIG. 20A. Liver enzymes were measured at one month.(FIGS. 51B and 51C) HFD-fed mice were treated with IL-22 Fc or controlIgG as in FIG. 47A. Liver enzymes (FIG. 51B) and abdominal fat pad (FIG.51C) were measured at one month. **P<0.01, ***P<0.001, Error bars,s.e.m. (FIGS. 51D-51H) mice were fed with HFD for 10 weeks, and thentreated with IL-22 Fc or control twice per week for 6 weeks. (FIG. 51D)Lipid metabolic gene expression from white adipose tissue. (FIG. 51E)Serum triglyceride, glycerol and free fatty acid. (FIG. 51F) Hepatictriglyceride. (FIG. 51G) Hepatic cholesterol. (FIG. 51H) White adiposetissue triglyceride. (FIGS. 51I and 51J) db/db mice treated with IL-22Fc or control IgG for 4 weeks. (FIG. 51I) Hepatic triglyceride. (FIG.51J) White adipose tissue triglyceride. *P<0.05. Error bars, s.e.m.

FIGS. 52A-52C show results demonstrating that IL-22 increased insulinsecretion of β cells. db/db mice were treated with IL-22 Fc as in FIG.20A, Pancreases were harvested on day 30 and stained for insulin andglucagon. (FIG. 52A) Percentage of islet area within total pancreasarea. (FIG. 52B) Percentage of β cell area within total islet area.(FIG. 52C) Percentage of a cell area within total islet area.

FIG. 53 IL-22 KO mice did not develop glucose intolerance with HFD.IL-22 KO mice were fed with HFD starting at 6 weeks of age. Glucosetolerance test was done 3 months after HFD. Error bars, s.e.m.

FIGS. 54A-54G show results demonstrating susceptibility of ob/ob mice toC. rodentium infection: (FIG. 54A) body weight and (FIG. 54B) survivalof ob/ob and lean mice (n=10) infected with C. rodentium; (FIGS. 54C and54D) colon histology of lean control (FIG. 54C) and ob/ob mice (FIG.54D) on day 8, showing epithelial hyperplasia, enterocyte shedding intothe gut lumen, bacterial colonies (arrows) and submucosal edema(vertical bar) (horizontal bar, 200 μm); (FIG. 54E) clinical scoredetermined by colon histology (n=5); and (FIGS. 54F and 54G) bacterialburden of ob/ob and lean control mice (n=5) in liver (FIG. 54F) andspleen (FIG. 54G) on day 8. *P<0.05, ** P<0.01, ***P<0.001. Error bars,s.e.m.

FIGS. 55A-55C show results of db/db mice treated with IL-22 Fc, IL-20 Fcor IL-24 Fc in (FIG. 55A) body weight, (FIG. 55B) serum glucose and(FIG. 55C) glucose tolerance test on day 20 of treatment.

FIGS. 56A and 56B show results comparing wound healing efficacy in db/dbmice treated with VEGF or IL-22 Fc.

FIGS. 57A-57E show cytokine or chemokine induction by IL-22 Fc inreconstituted epidermis.

FIG. 58 shows results comparing wound closure using a splinted woundmodel in wild type mice and db/db mice with or without S. aureusinfection.

FIGS. 59A and 59B show results comparing wound healing efficacy betweenVEGF and IL-22 Fc in a splinted infected wound model.

FIG. 60 shows results comparing wound healing efficacy between VEGF andIL-22 Fc at different concentrations in a splinted infected wound model.

FIG. 61 shows results comparing wound healing efficacy between VEGF,PDGF and IL-22 Fc at different concentrations in a splinted infectedwound model.

FIG. 62 shows that IL-22 Fc accelerated wound healing in a solution aswell as in a gel formulation in a splinted wound model.

DETAILED DESCRIPTION OF EMBODIMENTS OF THE INVENTION

All publications, patents and patent applications cited herein arehereby expressly incorporated by reference for all purposes.

In one aspect, the present invention concerns the IL-22 protein or IL-22Fc fusion proteins, composition comprising the same, and methods ofusing the same. In particular, the invention concerns using IL-22 Fcfusion proteins or IL-22 polypeptide in the prevention and treatment ofIBD, atherosclerosis, cardiovascular diseases and conditionscharacterized by atherosclerotic plaque formation, metabolic syndrome,mild and acute endotoxemia and sepsis, acute kidney injury, acutepancreatitis, moderate acute pancreatitis, and insulin-relateddisorders. Further, the invention concerns using IL-22 Fc fusionproteins or IL-22 polypeptides in the prevention and treatment ofdiabetic foot ulcer, accelerating wound healing and in particulardiabetic wound healing.

In one aspect, it is believed that this is the first disclosure showingIL-22 polypeptide treating cardiovascular disease per se. The dataherein supports the notion that an IL-22 polypeptide or IL-22 Fc fusionprotein can reduce the growth of atherosclerotic plaques, reduce thefrequency of rupture of atherosclerotic plaques and reduce endotoxemia.This invention is particularly useful in treating subjects sufferingfrom metabolic syndrome, mild or acute endotoxemia, sepsis andinsulin-related disorders, such as insulin-resistance (no responsive toinsulin) who need a change to their HDL/LDL lipid profile, as can bedetermined by a doctor or clinician. The application shows data thatindicate that IL-22 polypeptide or IL-22 Fc fusion protein can increasehigh density lipoproteins (HDL) and decrease low density lipoproteins(LDL) in those subjects suffering from metabolic syndrome. The data,without being bound by theory, also indicate gut-derived LPS a driverbehind endotoxemia and atherosclerosis. Mice treated with mIL-22 Fcfusion protein had reduced hyperlipidemia, improved glucose tolerancewith restored vascular function and these changes culminated in areduction in atherosclerotic plaque. IL-22 polypeptide or IL-22 Fcfusion protein can attenuate the progression of cardiovascular disease.

Further, diabetes is a chronic disorder affecting carbohydrate, fat andprotein metabolism in animals. Diabetes is the leading cause ofblindness, renal failure, and lower limb amputations in adults and is amajor risk factor for cardiovascular disease and stroke. Type I diabetesmellitus (or insulin-dependent diabetes mellitus (“IDDM”) orjuvenile-onset diabetes) comprises approximately 10% of all diabetescases. The disease is characterized by a progressive loss of insulinsecretory function by beta cells of the pancreas. This characteristic isalso shared by non-idiopathic, or “secondary”, diabetes having itsorigins in pancreatic disease. Type I diabetes mellitus is associatedwith the following clinical signs or symptoms, e.g., persistentlyelevated plasma glucose concentration or hyperglycemia; polyuria;polydipsia and/or hyperphagia; chronic microvascular complications suchas retinopathy, nephropathy and neuropathy; and macrovascularcomplications such as hyperlipidemia and hypertension which can lead toblindness, end-stage renal disease, limb amputation and myocardialinfarction.

Type II diabetes mellitus (non-insulin-dependent diabetes mellitus orNIDDM, also referred to as type II diabetes) is a metabolic disorder (ormetabolic syndrome) involving the dysregulation of glucose metabolismand impaired insulin sensitivity. Type II diabetes mellitus usuallydevelops in adulthood and is associated with the body's inability toutilize or make sufficient insulin. In addition to the insulinresistance observed in the target tissues, patients suffering from typeII diabetes mellitus have a relative insulin deficiency—that is,patients have lower than predicted insulin levels for a given plasmaglucose concentration. Type II diabetes mellitus is characterized by thefollowing clinical signs or symptoms, e.g., persistently elevated plasmaglucose concentration or hyperglycemia; polyuria; polydipsia and/orhyperphagia; chronic microvascular complications such as retinopathy,nephropathy and neuropathy; and macrovascular complications such ashyperlipidemia and hypertension which can lead to blindness, end-stagerenal disease, limb amputation and myocardial infarction.

I. Definitions

Unless otherwise defined, all terms of art, notations and otherscientific terminology used herein are intended to have the meaningscommonly understood by those of skill in the art to which this inventionpertains. In some cases, terms with commonly understood meanings aredefined herein for clarity and/or for ready reference, and the inclusionof such definitions herein should not necessarily be construed torepresent a substantial difference over what is generally understood inthe art.

Within this application, unless otherwise stated, the techniquesutilized may be found in any of several well-known references such as:Molecular Cloning: A Laboratory Manual (Sambrook, et al., 1989, ColdSpring Harbor Laboratory Press), PCR Protocols: A Guide to Methods andApplications (Innis, et al. 1990. Academic Press, San Diego, Calif.),and Harlow and Lane (1988) Antibodies: A Laboratory Manual ch. 14 (ColdSpring Harbor Laboratory, Cold Spring Harbor, N.Y.).

As appropriate, procedures involving the use of commercially availablekits and reagents are generally carried out in accordance withmanufacturer defined protocols and/or parameters unless otherwise noted.Before the present methods and uses therefore are described, it is to beunderstood that this invention is not limited to the particularmethodology, protocols, cell lines, animal species or genera,constructs, and reagents described as such can, of course, vary. It isalso to be understood that the terminology used herein is for thepurpose of describing particular embodiments only, and is not intendedto limit the scope of the present invention which will be limited onlyby the appended claims.

As used herein, the singular forms “a”, “an” and “the” include pluralreferents unless the context clearly dictates otherwise. For example,reference to “an isolated peptide” means one or more isolated peptides.

Throughout this specification and claims, the word “comprise,” orvariations such as “comprises” or “comprising,” will be understood toimply the inclusion of a stated integer or group of integers but not theexclusion of any other integer or group of integers.

The term “IL-22 Fc fusion protein” or “IL-22 fusion protein” or “IL-22Ig fusion protein” as used herein refers to a fusion protein in whichIL-22 protein or polypeptide is linked, directly or indirectly, to anIgG Fc region. In certain preferred embodiments, the IL-22 Fc fusionprotein of the invention comprises a human IL-22 protein or polypeptidelinked to a human IgG Fc region. In certain embodiments, the human IL-22protein comprises the amino acid sequence of SEQ ID NO:4. However, it isunderstood that minor sequence variations such as insertions, deletions,substitutions, especially conservative amino acid substitutions of IL-22or Fc that do not affect the function and/or activity of IL-22 or IL-22Fc fusion protein are also contemplated by the invention. The IL-22 Fcfusion protein of the invention can bind to IL-22 receptor, which canlead to IL-22 receptor downstream signaling. In certain embodiments, theIL-22 Fc fusion protein is capable of binding to IL-22 receptor, and/oris capable of leading to IL-22 receptor downstream signaling. Thefunctions and/or activities of the IL-22 Fc fusion protein can beassayed by methods known in the art, including without limitation,ELISA, ligand-receptor binding assay and Stat3 luciferase assay. Incertain embodiments, the invention provides an IL-22 Fc fusion proteinthat binds to IL-22 receptor, the binding can lead to IL-22 receptordownstream signaling, said IL-22 Fc fusion protein comprising an aminoacid sequence having at least 95% sequence identity to the amino acidsequence selected from the group consisting of SEQ ID NO:8, SEQ IDNO:10, SEQ ID NO:12 and SEQ ID NO:14, and wherein the Fc region is notglycosylated. In certain particular embodiments, the Fc region of theIL-22 fusion protein does not possess effector activities (e.g., doesnot bind to FcγIIIR) or exhibits substantially lower effector activitythan a whole (e.g., wild type) IgG antibody. In certain otherembodiments, the Fc region of the IL-22 Fc fusion protein does nottrigger cytotoxicity such as antibody-dependent cellular cytotoxicity(ADCC) or complement dependent cytotoxicity (CDC). Unless otherwisespecified, “IL-22 fusion protein,” “IL-22 Fc fusion,” “IL-22 Ig fusionprotein,” “IL-22 Fc fusion protein” or “IL-22 Fc” are usedinterchangeably throughout this application.

The term “IL-22” or “IL-22 polypeptide” or “IL-22 protein” as usedherein, broadly refers to any native IL-22 from any mammalian source,including primates (e.g. humans) and rodents (e.g., mice and rats),unless otherwise indicated. The term encompasses “full-length,”unprocessed IL-22 as well as any forms of IL-22 that result fromprocessing in the cell. For example, both full-length IL-22 containingthe N-terminal leader sequence and the mature form IL-22 are encompassedby the current invention. The leader sequence (or signal peptide) can bethe endogenous IL-22 leader sequence or an exogenous leader sequence ofanother mammalian secretary protein. In certain embodiments, the leadersequence can be from a eukaryotic or prokaryotic secretary protein. Theterm also encompasses naturally occurring variants of IL-22, e.g.,splice variants or allelic variants. The amino acid sequence of anexemplary human IL-22 is shown in SEQ ID NO:4 (mature form, without asignal peptide). In certain embodiments, the amino acid sequence offull-length IL-22 protein with the endogenous leader sequence isprovided in SEQ ID NO:71; while in other embodiments, the amino acidsequence of mature IL-22 protein with an exogenous leader sequence isprovided in SEQ ID NO:2. Minor sequence variations especiallyconservative amino acid substitutions of IL-22 that do not affect theIL-22's function and/or activity (e.g., binding to IL-22 receptor) arealso contemplated by the invention. FIG. 1 shows an amino acid sequencealignment of mature IL-22 from several exemplary mammalian species. Theasterisks indicate highly conserved amino acid residues across speciesthat are likely important for the functions and/or activities of IL-22.Accordingly, in certain embodiments, the IL-22 Fc fusion protein of theinvention comprises an IL-22 polypeptide comprising an amino acidsequence having at least 95%, at least 96%, at least 97%, at least 98%or at least 99% sequence identity to SEQ ID NO:4. In certain otherembodiments, the IL-22 protein has 95% or more sequence identity to SEQID NO:71, 96% or more sequence identity to SEQ ID NO:71, 97% or moresequence identity to SEQ ID NO:71; 98% or more sequence identity to SEQID NO:71; 99% or more sequence identity to SEQ ID NO:71. The IL-22polypeptides described herein can be isolated from a variety of sources,such as from human tissue or from another source, or prepared byrecombinant or synthetic methods.

The term “IL-22 receptor” or “IL-22R” refers to a heterodimer consistingof IL-22R1 and IL-10R2 or naturally occurring allelic variants thereof.See Ouyang et al., 2011, Annu. Rev. Immunol. 29:159-63. IL-10R2 isubiquitously expressed by many cell types, and IL-22R1 is expressed onlyin innate cells such as epithelial cells, hepatocytes and keratinocytes.IL-22R1 is also known as IL-22Ra1 or IL-22Ra1. IL-22R1 may be pairedwith other polypeptides to form heterodimeric receptors for other IL-10family members, for example IL-20 or IL-24. See e.g., Ouyang et al.,2011, supra.

A “native sequence IL-22 polypeptide” or a “native sequence IL-22Rpolypeptide” refers to a polypeptide comprising the same amino acidsequence as a corresponding IL-22 or IL-22R polypeptide derived fromnature. Such native sequence IL-22 or IL-22R polypeptides can beisolated from nature or can be produced by recombinant or syntheticmeans. The terms specifically encompass naturally-occurring truncated orsecreted forms of the specific IL-22 or IL-22R polypeptide (e.g., anIL-22 lacking its associated signal peptide), naturally-occurringvariant forms (e.g., alternatively spliced forms), andnaturally-occurring allelic variants of the polypeptide. In variousembodiments of the invention, the native sequence IL-22 or IL-22Rpolypeptides disclosed herein are mature or full-length native sequencepolypeptides. An exemplary full length native human IL-22 is shown inFIG. 30 (DNA, SEQ ID NO:70) and FIG. 31 (protein, SEQ ID NO:71). Thestart and stop codons are shown in bold font and underlined in FIG. 30.While the IL-22 and IL-22R polypeptide sequences disclosed in theaccompanying figures are shown to begin with methionine residuesdesignated herein as amino acid position 1, it is conceivable andpossible that other methionine residues located either upstream ordownstream from the amino acid position 1 in the figures can be employedas the starting amino acid residue for the IL-22 or IL-22R polypeptides.

An “IL-22 variant,” an “IL-22R variant,” an “IL-22 variant polypeptide,”or an “IL-22R variant polypeptide” means an active IL-22 or IL-22Rpolypeptide as defined above having at least about 80% amino acidsequence identity with a full-length native sequence IL-22 or IL-22Rpolypeptide sequence. Ordinarily, an IL-22 or IL-22R polypeptide variantwill have at least about 80% amino acid sequence identity, alternativelyat least about 81% amino acid sequence identity, alternatively at leastabout 82% amino acid sequence identity, alternatively at least about 83%amino acid sequence identity, alternatively at least about 84% aminoacid sequence identity, alternatively at least about 85% amino acidsequence identity, alternatively at least about 86% amino acid sequenceidentity, alternatively at least about 87% amino acid sequence identity,alternatively at least about 88% amino acid sequence identity,alternatively at least about 89% amino acid sequence identity,alternatively at least about 90% amino acid sequence identity,alternatively at least about 91% amino acid sequence identity,alternatively at least about 92% amino acid sequence identity,alternatively at least about 93% amino acid sequence identity,alternatively at least about 94% amino acid sequence identity,alternatively at least about 95% amino acid sequence identity,alternatively at least about 96% amino acid sequence identity,alternatively at least about 97% amino acid sequence identity,alternatively at least about 98% amino acid sequence identity, andalternatively at least about 99% amino acid sequence identity to afull-length or mature native sequence IL-22 or IL-22R polypeptidesequence.

The term “Fc region,” “Fc domain” or “Fc” refers to a C-terminalnon-antigen binding region of an immunoglobulin heavy chain thatcontains at least a portion of the constant region. The term includesnative Fc regions and variant Fc regions. In certain embodiments, ahuman IgG heavy chain Fc region extends from Cys226 to thecarboxyl-terminus of the heavy chain. However, the C-terminal lysine(Lys447) of the Fc region may or may not be present, without affectingthe structure or stability of the Fc region. Unless otherwise specifiedherein, numbering of amino acid residues in the IgG or Fc region isaccording to the EU numbering system for antibodies, also called the EUindex, as described in Kabat et al., Sequences of Proteins ofImmunological Interest, 5th Ed. Public Health Service, NationalInstitutes of Health, Bethesda, Md., 1991.

In certain embodiments, Fc region refers to an immunoglobulin IgG heavychain constant region comprising a hinge region (starting at Cys226), anIgG CH2 domain and CH3 domain. The term “hinge region” or “hingesequence” as used herein refers to the amino acid sequence locatedbetween the linker and the CH2 domain. In certain embodiments, the hingeregion comprises the amino acid sequence CPPCP (SEQ ID NO:31). Incertain embodiments, the hinge region for IL-22 IgG4 Fc fusion proteincomprises the CPPCP sequence (SEQ ID NO:31), a sequence found in thenative IgG1 hinge region, to facilitate dimerization. In certain otherembodiments, the Fc region starts at the hinge region and extends to theC-terminus of the IgG heavy chain. In certain particular embodiments,the Fc region comprises the Fc region of human IgG1, IgG2, IgG3 or IgG4.In certain particular embodiments, the Fc region comprises the CH2 andCH3 domain of IgG4. In certain other particular embodiments, the Fcregion comprises the CH2 and CH3 domain of IgG1. As described in theExample section, it was unexpectedly discovered by the applicants thatIL-22 IgG4 Fc fusion protein exhibited even superior pharmacokineticproperties than IL-22 IgG1 Fc fusion protein.

In certain embodiments, the IgG CH2 domain starts at Ala 231. In certainother embodiments, the CH3 domain starts at Gly 341. It is understoodthat the C-terminus Lys residue of human IgG can be optionally absent.It is also understood that conservative amino acid substitutions of theFc region without affecting the desired structure and/or stability of Fcis contemplated within the scope of the invention.

In certain embodiments, the IL-22 is linked to the Fc region via alinker. In certain particular embodiments, the linker is a peptide thatconnects the C-terminus of IL-22 to the Fc region as described herein.In certain embodiments, native IgG sequences are present in the linkerand/or hinge region to minimize and/or avoid the risk of immunogenicity.In other embodiments, minor sequence variations can be introduced to thenative sequences to facilitate manufacturing. IL-22 Fc fusion constructscomprising exogenous linker or hinge sequences that exhibit highactivity (as measured, e.g., by a luciferase assay) are also within thescope of the invention. In certain embodiments, the linker comprises anamino acid sequence that is 8-20 amino acids, 8-16, 8-15, 8-14, 8-13,8-12, 8-11, 8-10, 8-9, 10-11, 10-12, 10-13, 10-14, 10-15, 10-16, 11-16,8, 9, 10, 11, 12, 13, 14, 15 or 16 amino acids long. In certain otherembodiments, the linker comprises the amino acid sequence DKTHT (SEQ IDNO:32).

In certain particular embodiments, the linker does not comprise thesequence Gly-Gly-Ser (SEQ ID NO:45), Gly-Gly-Gly-Ser (SEQ ID NO:46) orGly-Gly-Gly-Gly-Ser (SEQ ID NO:47).

In certain embodiments, the IL-22 Fc fusion protein comprises an IL-22polypeptide linked to an Fc region by a linker. The term “linked to” or“fused to” refers to a covalent bond, e.g., a peptide bond, formedbetween two moieties.

The term “afucosylation,” “afucosylated,” “defucosylation,” or“defucosylated” refers to the absence or removal of core-fucose from theN-glycan attached to the CH2 domain of Fc.

It was unexpectedly discovered by the applicants that IL-22 IgG1 Fcfusion proteins, unlike other Fc fusion proteins or antibodiescomprising Fc, exhibited high levels (e.g., 30%) of afucosylation in theN-glycans attached to the Fc region. The N-glycans attached to the CH2domain of Fc is heterogeneous. Antibodies or Fc fusion proteinsgenerated in CHO cells are fucosylated by fucosyltransferase activity.See Shoji-Hosaka et al., J. Biochem. 2006, 140:777-83. Normally, a smallpercentage of naturally occurring afucosylated IgGs may be detected inhuman serum. N-glycosylation of the Fc is important for binding to FcγR;and afucosylation of the N-glycan increases Fc's binding capacity toFcγRIIIa. Increased FcγRIIIa binding can enhance antibody-dependentcellular cytotoxicity (ADCC), which can be advantageous in certainantibody therapeutic applications in which cytotoxicity is desirable.See Shoji-Hosaka et al., supra. Such an enhanced effector function,however, can be detrimental when Fc-mediated cytotoxicity is undesirablesuch as in the case of IL-22 Fc fusion.

IgG4 Fc is known to exhibit less effector activity than IgG1 Fc.Applicants unexpectedly discovered that IL-22 IgG4 Fc fusion proteinalso showed high levels of afucosylation in the Fc region. Thehigh-level of afucosylated N-glycan attached to the Fc of IgG4 canincrease the undesirable effector activity.

Thus, in one aspect, the invention provides an IL-22 Fc fusion proteinin which the Fc region or CH2 domain is not glycosylated. In certainembodiments, the N-glycosylation site in the CH2 domain is mutated toprevent from glycosylation.

In certain other embodiments, the glycosylation in the CH2 domain of theFc region can be eliminated by altering the glycosylation consensussite, i.e., Asn at position 297 followed by any amino acid residue (inthe case of human IgG, Ser) and Thr (see FIG. 3). The glycosylation sitecan be altered by amino acid insertions, deletions and/or substitutions.For example, one or more amino acid residues can be inserted between Asnand Ser or between Ser and Thr to alter the original glycosylation site,wherein the insertions do not regenerate an N-glycosylation site. Incertain particular embodiments, the N297 residue (e.g., theN-glycosylated site in Fc, see FIG. 3) within the CH2 domain of humanIgG Fc is mutated to abolish the glycosylation site. In certainparticular embodiments, the N297 residue is changed to Gly, Ala, Gln,Asp or Glu. In some particular embodiments, the N297 residue is changedto Gly or Ala. In other particular embodiments, the N297 residue ischanged to Gly. In certain other embodiments, the T299 residue can besubstituted with another amino acid, for example Ala, Val or Gly. Incertain particular embodiments, the mutations that result in anaglycosylated Fc do not affect the structure and/or stability of theIL-22 Fc fusion protein.

In a related aspect, the invention provides a method of treating IBD,including UC and CD, methods of inhibiting bacterial infection in theintestine, and methods of improving epithelial integrity, epithelialproliferation, differentiation and/or migration in the intestine, andmethods of treating metabolic disorders or metabolic syndrome, type IIdiabetes, atherosclerosis and diabetic wound healing in a patient inneed thereof comprising administering to the patient a pharmaceuticalcomposition comprising an IL-22 Fc fusion protein wherein the Fc regionis not glycosylated.

In a further aspect, the invention provides a composition comprisingIL-22 Fc fusion proteins having low level of or no afucosylation in theFc region. Specifically, the invention provides a composition comprisingIL-22 Fc fusion proteins having an overall afucosylation level in the Fcregion of no more than 10%, preferably no more than 5%, more preferablyno more than 2%, and most preferably less than 1%. In another aspect,the invention provides methods of treating IBD, including UC and CD,methods of inhibiting bacterial infection in the intestine, and methodsof improving epithelial integrity, epithelial proliferation,differentiation and/or migration in the intestine, and methods oftreating metabolic disorders, type II diabetes, type II diabetes withmorbid obesity, graft versus host disease (GVHD), atherosclerosis,cardiovascular disease, metabolic syndrome, endotoxemia (acute andmild), sepsis, acute coronary heart disease, hypertension, dyslipemia,obesity, hyperglycemia, lipid metabolism disorders, hepatitis, acutehepatitis, renal failure, acute renal failure, acute kidney injury,rental draft failure, pancreatitis, acute pancreatitis, liver fibrosisand lung fibrosis, wound, infected wound, accelerating wound healing,including diabetic wound healing, in a patient in need thereofcomprising administering to the patient a pharmaceutical compositioncomprising IL-22 Fc fusion proteins having an afucosylation level in theFc region of no more than 10%, preferably no more than 5%, morepreferably no more than 2%, and most preferably less than 1%.

The term “% afucosylation” refers to the level of afucosylation in theFc region in a composition of IL-22 Fc fusion proteins. The %afucosylation can be measured by mass spectrometry (MS) and presented asthe percentage of afucosylated glycan species (species without thefucose on one Fc domain (minus 1) and on both Fc domains (minus 2)combined) over the entire population of IL-22 Fc fusion proteins. Forexample, % afucosylation can be calculated as the percentage of thecombined area under the minus 1 fucose peak and minus 2 fucose peak overthe total area of all glycan species analyzed by MS, such as determinedby an Agilent 6520B TOF Mass Spectrometer as described in FIG. 2 and inthe examples shown below. The level of afucosylation can be measured byany other suitable methods known in the art, including withoutlimitation HPLC-Chip Cube MS (Agilent) and reverse phase-HPLC. The %afucosylation of IL-22 Fc composition can be used as an indication fordetermining whether the composition will likely trigger unacceptablelevel of ADCC, unsuitable for the intended purposes. Accordingly, incertain particular embodiments, the composition comprises IL-22 Fcfusion proteins having an afucosylation level of no more than 10%,preferably no more than 5%, more preferably no more than 3%, and mostpreferably no more than 1%. In certain embodiments, the compositioncomprises IL-22 Fc fusion proteins having an afucosylation level of nomore than 10%, no more than 9%, no more than 8%, no more than 7%, nomore than 6%, no more than 5%, no more than 4%, no more than 3%, no morethan 2%, or no more than 1%.

In certain embodiments, the desired level of afucosylation of an IL-22Fc composition can be achieved by methods known in the art, includingwithout limitation, by purification. For example, the fucosylatedspecies in a composition can be enriched by affinity chromatographyhaving resins conjugated with a fucose binding moiety, such as anantibody or lectin specific for fucose, especially fucose present in the1-6 linkage. See e.g., Kobayashi et al, 2012, J. Biol. Chem.287:33973-82. In certain other embodiments, the fucosylated species canbe enriched and separated from afucosylated species using an anti-fucosespecific antibody in an affinity column. Alternatively or additionally,afucosylated species can be separated from fucosylated species based onthe differential binding affinity to FcγRIIIa using affinitychromatography.

In certain other embodiments, the IL-22 Fc fusion protein comprises anFc region in which the N297 residue in the CH2 domain is mutated. Incertain embodiments, the N297 residue is changed to Gly or Ala,preferably to Gly. In certain other embodiments, the N297 residue isdeleted. In certain embodiments, the IL-22 Fc fusion protein comprisingan Fc having an amino acid substitution at N297 is aglycosylated or notglycosylated. The term “aglycosylated” as used herein refers to aprotein or a portion of a protein of interest that is not glycosylated.For example, an IL-22 Fc fusion protein with an aglycosylated Fc regioncan be made by mutagenizing the N297 residue in the CH2 domain of the Fcregion.

In other embodiments, the N-glycan attached to the wild type N297residue can be removed enzymatically, e.g., by deglycosylation. Suitableglycolytic enzymes include without limitation, peptide-N-glycosidase(PNGase).

The term “dimeric IL-22 Fc fusion protein” refers to a dimer in whicheach monomer comprises an IL-22 Fc fusion protein. The term “monomericIL-22 Fc fusion protein” refers to a dimer in which one monomercomprises an IL-22 Fc fusion protein (the IL-22 Fc arm), while the othermonomer comprises an Fc region without the IL-22 polypeptide (the Fcarm). Accordingly, the dimeric IL-22 Fc fusion protein is bivalent withrespect to IL-22R binding, whereas the monomeric IL-22 Fc fusion proteinis monovalent with respect to IL-22R binding. The heterodimerization ofthe monomeric IL-22 Fc fusion protein can be facilitated by methodsknown in the art, including without limitation, heterodimerization bythe knob-into-hole technology. The structure and assembly method of theknob-into-hole technology can be found in, e.g., U.S. Pat. Nos.5,821,333, 7,642,228, US 2011/0287009 and PCT/US2012/059810, herebyincorporated by reference in their entireties. This technology wasdeveloped by introducing a “knob” (or a protuberance) by replacing asmall amino acid residue with a large one in the CH3 domain of one Fc,and introducing a “hole” (or a cavity) in the CH3 domain of the other Fcby replacing one or more large amino acid residues with smaller ones. Incertain embodiments, the IL-22 Fc fusion arm comprises a knob, and theFc only arm comprises a hole.

The preferred residues for the formation of a knob are generallynaturally occurring amino acid residues and are preferably selected fromarginine (R), phenylalanine (F), tyrosine (Y) and tryptophan (W). Mostpreferred are tryptophan and tyrosine. In one embodiment, the originalresidue for the formation of the knob has a small side chain volume,such as alanine, asparagine, aspartic acid, glycine, serine, threonineor valine. Exemplary amino acid substitutions in the CH3 domain forforming the knob include without limitation the T366W, T366Y or F405Wsubstitution.

The preferred residues for the formation of a hole are usually naturallyoccurring amino acid residues and are preferably selected from alanine(A), serine (S), threonine (T) and valine (V). In one embodiment, theoriginal residue for the formation of the hole has a large side chainvolume, such as tyrosine, arginine, phenylalanine or tryptophan.Exemplary amino acid substitutions in the CH3 domain for generating thehole include without limitation the T366S, L368A, F405A, Y407A, Y407Tand Y407V substitutions. In certain embodiments, the knob comprisesT366W substitution, and the hole comprises the T366S/L368A/Y407Vsubstitutions. In certain particular embodiments, the Fc region of themonomeric IL-22 Fc fusion protein comprises an IgG1 Fc region. Incertain particular embodiments, the monomeric IL-22 IgG1 Fc fusioncomprises an IL-22 Fc knob arm and an Fc hole arm. In certainembodiments, the IL-22 Fc knob arm comprises a T366W substitution (SEQID NO:61), and the Fc hole arm comprises T366S, L368A and Y407V (SEQ IDNO:62). In certain other embodiments, the Fc region of both arms furthercomprises an N297G or N297A mutation. In certain embodiments, themonomeric IL-22 Fc fusion protein is expressed in E. coli cells. It isunderstood that other modifications to the Fc region known in the artthat facilitate heterodimerization are also contemplated and encompassedby the instant application.

The term “wound” refers to an injury, especially one in which the skinor another external surface is torn, pierced, cut, or otherwise broken.

The term “ulcer” is a site of damage to the skin or mucous membrane thatis often characterized by the formation of pus, death of tissue, and isfrequently accompanied by an inflammatory reaction.

The term “intestine” or “gut” as used herein broadly encompasses thesmall intestine and large intestine.

The term “accelerating wound healing” or “acceleration of wound healing”refers to the increase in the rate of healing, e.g., a reduction in timeuntil complete wound closure occurs or a reduction in time until a %reduction in wound area occurs.

A “diabetic wound” is a wound that associated with diabetes.

A “diabetic ulcer” is an ulcer that is associated with diabetes.

A “chronic wound” refers to a wound that does not heal. See, e.g.,Lazarus et al., Definitions and guidelines for assessment of wounds andevaluation of healing, Arch. Dermatol. 130:489-93 (1994). Chronic woundsinclude, but are not limited to, e.g., arterial ulcers, diabetic ulcers,pressure ulcers or bed sores, venous ulcers, etc. An acute wound candevelop into a chronic wound. Acute wounds include, but are not limitedto, wounds caused by, e.g., thermal injury (e.g., burn), trauma,surgery, excision of extensive skin cancer, deep fungal and bacterialinfections, vasculitis, scleroderma, pemphigus, toxic epidermalnecrolysis, etc. See, e.g., Buford, Wound Healing and Pressure Sores,HealingWell.com, published on: Oct. 24, 2001. Thus, in certainembodiments, a chronic wound is an infected wound. A “normal wound”refers to a wound that undergoes normal wound healing repair.

An “acceptor human framework” for the purposes herein is a frameworkcomprising the amino acid sequence of a light chain variable domain (VL)framework or a heavy chain variable domain (VH) framework derived from ahuman immunoglobulin framework or a human consensus framework, asdefined below. An acceptor human framework “derived from” a humanimmunoglobulin framework or a human consensus framework may comprise thesame amino acid sequence thereof, or it may contain amino acid sequencechanges. In some embodiments, the number of amino acid changes are 10 orless, 9 or less, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less,3 or less, or 2 or less. In some embodiments, the VL acceptor humanframework is identical in sequence to the VL human immunoglobulinframework sequence or human consensus framework sequence.

“Affinity” refers to the strength of the sum total of non-covalentinteractions between a single binding site of a molecule (e.g., a ligandor an antibody) and its binding partner (e.g., a receptor or anantigen). Unless indicated otherwise, as used herein, “binding affinity”refers to intrinsic binding affinity which reflects a 1:1 interactionbetween members of a binding pair (e.g., IL-22 Fc fusion protein andIL-22 receptor). The affinity of a molecule X for its partner Y cangenerally be represented by the dissociation constant (Kd). Affinity canbe measured by common methods known in the art, including thosedescribed herein. Specific illustrative and exemplary embodiments formeasuring binding affinity are described in the following.

The term “antibody” herein is used in the broadest sense and encompassesvarious antibody structures, including but not limited to monoclonalantibodies, polyclonal antibodies, multispecific antibodies (e.g.,bispecific antibodies), and antibody fragments so long as they exhibitthe desired antigen-binding activity.

An “antibody fragment” refers to a molecule other than an intactantibody that comprises a portion of an intact antibody that binds theantigen to which the intact antibody binds. Examples of antibodyfragments include but are not limited to Fv, Fab, Fab′, Fab′-SH,F(ab′)₂; diabodies; linear antibodies; single-chain antibody molecules(e.g. scFv); and multispecific antibodies formed from antibodyfragments.

An “antibody that binds to the same epitope” as a reference antibodyrefers to an antibody that blocks binding of the reference antibody toits antigen in a competition assay by 50% or more, and conversely, thereference antibody blocks binding of the antibody to its antigen in acompetition assay by 50% or more. An exemplary competition assay isprovided herein.

The term “chimeric” antibody refers to an antibody in which a portion ofthe heavy and/or light chain is derived from a particular source orspecies, while the remainder of the heavy and/or light chain is derivedfrom a different source or species.

The “class” of an antibody refers to the type of constant domain orconstant region possessed by its heavy chain. There are five majorclasses of antibodies: IgA, IgD, IgE, IgG, and IgM, and several of thesemay be further divided into subclasses (isotypes), e.g., IgG₁, IgG₂,IgG₃, IgG₄, IgA₁, and IgA₂. The heavy chain constant domains thatcorrespond to the different classes of immunoglobulins are called α, δ,ε, γ, and μ, respectively.

The term “cytotoxic agent” as used herein refers to a substance thatinhibits or prevents a cellular function and/or causes cell death ordestruction. Cytotoxic agents include, but are not limited to,radioactive isotopes (e.g., At²¹¹, I¹³¹, I¹²⁵, Y⁹⁰, Re¹⁸⁶, Re¹⁸⁸, Sm¹⁵³,Bi²¹², P³², Pb²¹² and radioactive isotopes of Lu); chemotherapeuticagents or drugs (e.g., methotrexate, adriamicin, vinca alkaloids(vincristine, vinblastine, etoposide), doxorubicin, melphalan, mitomycinC, chlorambucil, daunorubicin or other intercalating agents); growthinhibitory agents; enzymes and fragments thereof such as nucleolyticenzymes; antibiotics; toxins such as small molecule toxins orenzymatically active toxins of bacterial, fungal, plant or animalorigin, including fragments and/or variants thereof; and the variousantitumor or anticancer agents disclosed below.

“Effector functions” or “effector activities” refer to those biologicalactivities attributable to the Fc region of an antibody, which vary withthe antibody isotype. Examples of antibody effector functions include:C1q binding and complement dependent cytotoxicity (CDC); Fc receptorbinding; antibody-dependent cell-mediated cytotoxicity (ADCC);phagocytosis; down regulation of cell surface receptors (e.g. B cellreceptor); and B cell activation. In certain embodiments, the IL-22 Fcfusion protein does not exhibit any effector function or any detectableeffector function. In certain other embodiments, the IL-22 Fc fusionprotein exhibits substantially reduced effector function, e.g., about50%, 60%, 70% 80%, or 90% reduced effector function.

An “effective amount” or “therapeutically effective amount” of an agent,e.g., a pharmaceutical formulation, refers to an amount effective, atdosages and for periods of time necessary, to achieve the desiredtherapeutic or prophylactic result.

For example, in the case of a cardiovascular disease or condition, thetherapeutically effective amount of the IL-22 polypeptide, fusionprotein or agonist can reduce the degree of atherosclerotic plaqueformation; reduce the size of the atherosclerotic plaque(s); inhibit(i.e., slow to some extent and preferably stop) atherosclerotic plaque;inhibit (i.e., slow to some extent and preferably stop) thrombosis orrupture of an atherosclerotic plaque; and/or relieve to some extent oneor more of the symptoms associated with the disease or condition.

By “reduce or inhibit” is meant the ability to cause an overall decreasepreferably of 20% or greater, more preferably of 50% or greater, andmost preferably of 75%, 85%, 90%, 95%, or greater. Reduce or inhibit canrefer to the symptoms of the disorder being treated, the presence orsize of atherosclerotic plaques, or the number of atheroscleroticplaque(s).

A “suboptimal amount” refers to the amount less than the optimal amountof a therapeutic agent typically used for a certain treatment. When twotherapeutic agents are given to a subject, either concurrently orsequentially, each therapeutic agent can be given at a suboptimal amountas compared to the treatment when each therapeutic agent is given alone.For example, in certain embodiments, the subject in need of IBDtreatment is administered with the pharmaceutical composition comprisingthe IL-22 Fc fusion protein of the invention and a dexamethasone at asuboptimal amount.

“Framework” or “FR” refers to variable domain residues other thanhypervariable region (HVR) residues. The FR of a variable domaingenerally consists of four FR domains: FR1, FR2, FR3, and FR4.Accordingly, the HVR and FR sequences generally appear in the followingsequence in VH (or VL): FR1-H1(L1)-FR2-H2(L2)-FR3-H3(L3)-FR4.

The terms “full length antibody,” “intact antibody,” and “wholeantibody” are used herein interchangeably to refer to an antibody havinga structure substantially similar to a native antibody structure orhaving heavy chains that contain an Fc region as defined herein.

The terms “host cell,” “host cell line,” and “host cell culture” areused interchangeably and refer to cells into which exogenous nucleicacid has been introduced, including the progeny of such cells. Hostcells include “transformants” and “transformed cells,” which include theprimary transformed cell and progeny derived therefrom without regard tothe number of passages. The transformed cell includes transiently orstably transformed cell. Progeny may not be completely identical innucleic acid content to a parent cell, but may contain mutations. Mutantprogeny that have the same function or biological activity as screenedor selected for in the originally transformed cell are included herein.In certain embodiments, the host cell is transiently transfected withthe exogenous nucleic acid. In certain other embodiments, the host cellis stably transfected with the exogenous nucleic acid.

A “human antibody” is one which possesses an amino acid sequence whichcorresponds to that of an antibody produced by a human or a human cellor derived from a non-human source that utilizes human antibodyrepertoires or other human antibody-encoding sequences. This definitionof a human antibody specifically excludes a humanized antibodycomprising non-human antigen-binding residues.

A “human consensus framework” is a framework which represents the mostcommonly occurring amino acid residues in a selection of humanimmunoglobulin VL or VH framework sequences. Generally, the selection ofhuman immunoglobulin VL or VH sequences is from a subgroup of variabledomain sequences. Generally, the subgroup of sequences is a subgroup asin Kabat et al., Sequences of Proteins of Immunological Interest, FifthEdition, NIH Publication 91-3242, Bethesda Md. (1991), vols. 1-3. In oneembodiment, for the VL, the subgroup is subgroup kappa I as in Kabat etal., supra. In one embodiment, for the VH, the subgroup is subgroup IIIas in Kabat et al., supra.

A “humanized” antibody refers to a chimeric antibody comprising aminoacid residues from non-human HVRs and amino acid residues from humanFRs. In certain embodiments, a humanized antibody will comprisesubstantially all of at least one, and typically two, variable domains,in which all or substantially all of the HVRs (e.g., CDRs) correspond tothose of a non-human antibody, and all or substantially all of the FRscorrespond to those of a human antibody. A humanized antibody optionallymay comprise at least a portion of an antibody constant region derivedfrom a human antibody. A “humanized form” of an antibody, e.g., anon-human antibody, refers to an antibody that has undergonehumanization.

The term “hypervariable region” or “HVR” as used herein refers to eachof the regions of an antibody variable domain which are hypervariable insequence (“complementarity determining regions” or “CDRs”) and/or formstructurally defined loops (“hypervariable loops”) and/or contain theantigen-contacting residues (“antigen contacts”). Generally, antibodiescomprise six HVRs: three in the VH (H1, H2, H3), and three in the VL(L1, L2, L3). Exemplary HVRs herein include:

(a) hypervariable loops occurring at amino acid residues 26-32 (L1),50-52 (L2), 91-96 (L3), 26-32 (H1), 53-55 (H2), and 96-101 (H3) (Chothiaand Lesk, J. Mol. Biol. 196:901-917 (1987));

(b) CDRs occurring at amino acid residues 24-34 (L1), 50-56 (L2), 89-97(L3), 31-35b (H1), 50-65 (H2), and 95-102 (H3) (Kabat et al., Sequencesof Proteins of Immunological Interest, 5th Ed. Public Health Service,National Institutes of Health, Bethesda, Md. (1991));

(c) antigen contacts occurring at amino acid residues 27c-36 (L1), 46-55(L2), 89-96 (L3), 30-35b (H1), 47-58 (H2), and 93-101 (H3) (MacCallum etal. J. Mol. Biol. 262: 732-745 (1996)); and

(d) combinations of (a), (b), and/or (c), including HVR amino acidresidues 46-56 (L2), 47-56 (L2), 48-56 (L2), 49-56 (L2), 26-35 (H1),26-35b (H1), 49-65 (H2), 93-102 (H3), and 94-102 (H3).

Unless otherwise indicated, HVR residues and other residues in thevariable domain (e.g., FR residues) are numbered herein according toKabat et al., supra.

An “immunoconjugate” is an antibody or a fragment of an antibodyconjugated to one or more heterologous molecule(s), including but notlimited to a cytotoxic agent.

An “individual,” “subject” or “patient” is a mammal. Mammals include,but are not limited to, domesticated animals (e.g., cows, sheep, cats,dogs, and horses), primates (e.g., humans and non-human primates such asmonkeys), rabbits, and rodents (e.g., mice and rats). In certainembodiments, the individual, subject or patient is a human.

An “isolated” IL-22 fusion protein is one which has been separated fromthe environment of a host cell that recombinantly produces the fusionprotein. In some embodiments, an IL-22 fusion protein is purified togreater than 95% or 99% purity as determined by, for example,electrophoretic (e.g., SDS-PAGE, isoelectric focusing (IEF), capillaryelectrophoresis) or chromatographic (e.g., ion exchange or reverse phaseHPLC).

An “isolated” nucleic acid refers to a nucleic acid molecule that hasbeen separated from a component of its natural environment. An isolatednucleic acid includes a nucleic acid molecule contained in cells thatordinarily contain the nucleic acid molecule, but the nucleic acidmolecule is present extrachromosomally or at a chromosomal location thatis different from its natural chromosomal location.

“Isolated nucleic acid encoding IL-22 Fc fusion protein” refers to oneor more nucleic acid molecules encoding the IL-22 Fc fusion protein,including such nucleic acid molecule(s) in a single vector or separatevectors, such nucleic acid molecule(s) transiently or stably transfectedinto a host cell and such nucleic acid molecule(s) present at one ormore locations in a host cell.

The term “control sequences” refers to DNA sequences necessary for theexpression of an operably linked coding sequence in a particular hostorganism. The control sequences that are suitable for prokaryotes, forexample, include a promoter, optionally an operator sequence, and aribosome binding site. Eukaryotic cells are known to utilize promoters,polyadenylation signals, and enhancers.

Nucleic acid is “operably linked” when it is placed into a functionalrelationship with another nucleic acid sequence. For example, DNA for apresequence or secretory leader is operably linked to DNA for apolypeptide if it is expressed as a preprotein that participates in thesecretion of the polypeptide; a promoter or enhancer is operably linkedto a coding sequence if it affects the transcription of the sequence; ora ribosome binding site is operably linked to a coding sequence if it ispositioned so as to facilitate translation. Generally, “operably linked”means that the DNA sequences being linked are contiguous, and, in thecase of a secretory leader, contiguous and in reading phase. However,enhancers do not have to be contiguous. Linking is accomplished byligation at convenient restriction sites. If such sites do not exist,the synthetic oligonucleotide adaptors or linkers are used in accordancewith conventional practice.

The term “monoclonal antibody” as used herein refers to an antibodyobtained from a population of substantially homogeneous antibodies,i.e., the individual antibodies comprising the population are identicaland/or bind the same epitope, except for possible variant antibodies,e.g., containing naturally occurring mutations or arising duringproduction of a monoclonal antibody preparation, such variants generallybeing present in minor amounts. In contrast to polyclonal antibodypreparations, which typically include different antibodies directedagainst different determinants (epitopes), each monoclonal antibody of amonoclonal antibody preparation is directed against a single determinanton an antigen. Thus, the modifier “monoclonal” indicates the characterof the antibody as being obtained from a substantially homogeneouspopulation of antibodies, and is not to be construed as requiringproduction of the antibody by any particular method. For example, themonoclonal antibodies to be used in accordance with the presentinvention may be made by a variety of techniques, including but notlimited to the hybridoma method, recombinant DNA methods, phage-displaymethods, and methods utilizing transgenic animals containing all or partof the human immunoglobulin loci, such methods and other exemplarymethods for making monoclonal antibodies being described herein.

A “naked antibody” refers to an antibody that is not conjugated to aheterologous moiety (e.g., a cytotoxic moiety) or radiolabel. The nakedantibody may be present in a pharmaceutical formulation.

“Native antibodies” refer to naturally occurring immunoglobulinmolecules with varying structures. For example, native IgG antibodiesare heterotetrameric glycoproteins of about 150,000 daltons, composed oftwo identical light chains and two identical heavy chains that aredisulfide-bonded. From N- to C-terminus, each heavy chain has a variableregion (VH), also called a variable heavy domain or a heavy chainvariable domain, followed by three constant domains (CH1, CH2, and CH3).Similarly, from N- to C-terminus, each light chain has a variable region(VL), also called a variable light domain or a light chain variabledomain, followed by a constant light (CL) domain. The light chain of anantibody may be assigned to one of two types, called kappa (κ) andlambda (λ), based on the amino acid sequence of its constant domain.

A “native sequence Fc region” comprises an amino acid sequence identicalto the amino acid sequence of an Fc region found in nature. Nativesequence human Fc regions include, without limitation, a native sequencehuman IgG1 Fc region (non-A and A allotypes); native sequence human IgG2Fc region; native sequence human IgG3 Fc region; and native sequencehuman IgG4 Fc region, as well as naturally occurring variants thereof.

A “variant Fc region” comprises an amino acid sequence which differsfrom that of a native sequence Fc region by virtue of at least one aminoacid modification, preferably one or more amino acid substitution(s).Preferably, the variant Fc region has at least one amino acidsubstitution compared to a native sequence Fc region or to the Fc regionof a parent polypeptide, e.g. from about one to about ten amino acidsubstitutions, and preferably from about one to about five amino acidsubstitutions in a native sequence Fc region or in the Fc region of theparent polypeptide. The variant Fc region herein will preferably possessat least about 80% homology with a native sequence Fc region and/or withan Fc region of a parent polypeptide, and most preferably at least about90% homology therewith, more preferably at least about 95% homologytherewith. In certain embodiments, the variant Fc region is notglycosylated.

The term “inflammatory bowel disorder,” “inflammatory bowel disease” orIBD is used herein in the broadest sense and includes all diseases andpathological conditions the pathogenesis of which involves recurrentinflammation in the intestine, including small intestine and colon.Commonly seen IBD includes ulcerative colitis and Crohn's disease. IBDis not limited to UC and CD. The manifestations of the disease includebut not limited to inflammation and a decrease in epithelial integrityin the intestine.

The term “cardiovascular disease” or “cardiovascular disorder” is usedherein in the broadest sense and includes all diseases and pathologicalconditions the pathogenesis of which involves abnormalities of the bloodvessels, such as, for example, atherosclerotic plaque formation(including stable or unstable/vulnerable plaques), atherosclerosis,arteriosclerosis, arteriolosclerosis, and elevated systemiclipopolysaccharide (LPS) exposure. The term additionally includesdiseases and pathological conditions that benefit from the inhibition ofthe formation of atherosclerotic plaques. Cardiovascular diseasesinclude, without limitation, coronary artery atherosclerosis, coronarymicrovascular disease, stroke, carotid artery disease, peripheralarterial disease, ischemia, coronary artery disease (CAD), acutecoronary syndrome (ACS), coronary heart disease (CHD), conditionsassociated with CAD and CHD, cerebrovascular disease, peripheralvascular disease, aneurysm, vasculitis, venous thrombosis, diabetesmellitus, and metabolic syndromechronic kidney disease, remote tissueinjury after ischemia and reperfusion, cardiopulmonary bypass.Specifically included within this group are all cardiovascular diseasesassociated with the occurrence, development, or progression of which canbe controlled by the inhibition of the atherosclerotic plaque formation.

The term “cardiovascular condition” is used herein in the broadest senseand includes all cardiovascular conditions and diseases the pathology ofwhich involves atherosclerotic plaque formation (including stable orunstable/vulnerable plaques), atherosclerosis, arteriosclerosis,arteriolosclerosis, and elevated systemic lipopolysaccharide (LPS)exposure. Specifically included within this group are all cardiovascularconditions and diseases associated with the atherosclerotic plaqueformation, the occurrence, development, or progression of which can becontrolled by the inhibition of the atherosclerotic plaque formation.The term specifically includes diseases and pathological conditions thatbenefit from the inhibition of the formation of atherosclerotic plaques.Cardiovascular conditions include, without limitation, coronary arteryatherosclerosis, coronary microvascular disease, stroke, carotid arterydisease, peripheral arterial disease, ischemia, coronary artery disease(CAD), coronary heart disease (CHD), conditions associated with CAD andCHD, cerebrovascular disease and conditions associated withcerebrovascular disease, peripheral vascular disease and conditionsassociated with peripheral vascular disease, aneurysm, vasculitis,venous thrombosis, diabetes mellitus, and metabolic syndromechronickidney disease, remote tissue injury after ischemia and reperfusion, andcardiopulmonary bypass. “Conditions associated with cerebrovasculardisease” as used herein include, for example, transient ischemic attack(TIA) and stroke. “Conditions associated with peripheral vasculardisease” as used herein include, for example, claudication. Specificallyincluded within this group are all cardiovascular diseases andconditions associated with the occurrence, development, or progressionof which can be controlled by the inhibition of the atherosclerosticplaque formation.

Atherosclerotic plaque formation can occur as a result of an innateimmune response to metabolic endotoxemia, which is characterized byelevated levels of systemic lipopolysaccharides (LPS) that originatefrom gut microbiota and a loss of functional integrity in the gutmucosal barrier. The innate immune response to endotoxemia results inthe low-grade chronic inflammation that is responsible for plaqueformation.

The term “metabolic syndrome” is used herein in the broadest sense.Metabolic syndrome includes the co-occurrence in an adult subject ofseveral metabolic risk factors, including at least three of thefollowing five traits: abdominal obesity, which can be, for example, awaist circumference in men of greater than or equal to 90 cm and inwomen greater than or equal to 80 cm; elevated serum triglycerides,which can be, for example, greater than or equal to 150 mg/dL, or drugtreatment for elevated triglycerides; reduced serum HDL cholesterollevel, which can be, for example, below 40 mg/dL in men and below 50mg/dL in women, or drug treatment for low HDL cholesterol; hypertension,which can be, for example, systolic blood pressure greater than 130 mmHgand diastolic blood pressure greater than 85 mmHg, or drug treatment forhypertension; and elevated fasting plasma glucose, which can be, forexample, greater than or equal to 100 mg/dL, drug treatment for elevatedglucose, or previously diagnosed type 2 diabetes. See also Meigs, theMetabolic Syndrome (Insulin Resistance Syndrome or Syndrome X),http://www.uptodate.com/contents/the-metabolic-syndrome-insulin-resistance-syndrome-or-syndrome-x,the disclosure of which is hereby incorporated by reference herein.

For children over 16 years old, the above criteria for adults can beused. For children between 10-16 year old, metabolic syndrome includesthe co-occurrence in a subject of several metabolic risk factors,including at least three of the following five traits: abdominalobesity, which can be, for example, a waist circumference greater than90^(th) percentile; elevated serum triglycerides, which can be, forexample, greater than or equal to 110 mg/dL, greater than 95^(th)percentile, or drug treatment for elevated triglycerides; reduced serumHDL cholesterol level, which can be, for example, below 40 mg/dL, lessthan 5^(th) percentile, or drug treatment for low HDL cholesterol;hypertension, which can be, for example, systolic blood pressure greaterthan 130 mmHg and diastolic blood pressure greater than 85 mmHg, greaterthan 90^(th) percentile, or drug treatment for hypertension; andelevated fasting plasma glucose, which can be, for example, greater thanor equal to 100 mg/dL, impaired glucose tolerance, drug treatment forelevated glucose, or previously diagnosed type 2 diabetes.

Generally speaking, the risk factors that co-occur in metabolic syndromeinclude obesity (such as abdominal obesity), hyperglycemia,dyslipidemia, insulin resistance, and/or hypertension. All these riskfactors promote the development of atherosclerotic cardiovasculardisease, diabetes, or both. Metabolic syndrome can also feature chronicadipose tissue inflammation.

Metabolic syndrome can be recognized as a proinflammatory, prothrombicstate, and can be associated with elevated levels of one or more ofC-reactive protein, IL-6, LPS, and plasminogen activator inhibitor 1;such markers can be associated with an increased risk for subsequentdevelopment of atherosclerotic cardiovascular disease, diabetes, orboth.

Metabolic syndrome can be associated with several obesity-relateddisorders, including one or more of fatty liver disease with steatosis,fibrosis, and cirrhosis, hepatocellular and intrahepaticcholangiocarcinoma, chronic kidney disease, polycystic ovary syndrome,sleep disordered breathing, including obstructive sleep apnea, andhyperuricemia and gout.

The term “insulin-related disorder” encompasses diseases or conditionscharacterized by impaired glucose tolerance. In one embodiment, theinsulin-related disorder is diabetes mellitus including, withoutlimitation, Type I (insulin-dependent diabetes mellitus or IDDM), TypeII (non-insulin dependent diabetes mellitus or NIDDM) diabetes,gestational diabetes, and any other disorder that would be benefited byagents that stimulate insulin secretion. In another embodiment, theinsulin-related disorder is characterized by insulin resistance.

The term “sepsis” is used in its broadest sense and can encompass asystemic inflammatory state caused by severe infection. Sepsis cancaused by the immune system's response to a serious infection, mostcommonly bacteria, but also fungi, viruses, and parasites in the blood,urinary tract, lungs, skin, or other tissues.

The term “acute endotoxemia” is used in its broadest sense and canencompass the condition of increased plasma bacterial lipopolysaccharide(LPS). Acute endotoxemia in turn could result in sepsis. Increased LPSin systemic circulation will induce low grade chronic inflammation,activating the endogenous protective host response to elevate plasmalipids that, in the chronic condition contributes to diet inducedobesity, insulin resistance and atherosclerosis, and eventual CVDevents.

As used herein, “treatment” (and grammatical variations thereof such as“treat” or “treating”) refers to clinical intervention in an attempt toalter the natural course of the individual being treated, and can beperformed either for prophylaxis or during the course of clinicalpathology. Desirable effects of treatment include, but are not limitedto, preventing occurrence or recurrence of disease, alleviation ofsymptoms, diminishment of any direct or indirect pathologicalconsequences of the disease, preventing metastasis, decreasing the rateof disease progression, amelioration or palliation of the disease state,and remission or improved prognosis.

For example, with regard to IBD, “treatment” can refer to a decrease inthe likelihood of developing IBD, a decrease in the rate of developingIBD and a decrease in the severity of the disease. As another example,with regard to atherosclerotic plaque formation, “treatment” can referto a decrease in the likelihood of developing atherosclerotic plaquedeposits, a decrease in the rate of development of deposits, a decreasein the number or size of existing deposits, or improved plaquestability. Those in need of treatment include those already with thedisorder as well as those in which the disorder is to be prevented.Desirable effects of treatment include, but are not limited to,preventing occurrence or recurrence of disease, alleviating symptoms,diminishing any direct or indirect pathological consequences of thedisease, preventing the disease, decreasing the rate of diseaseprogression, ameliorating or palliating the disease state, and causingremission or improved prognosis. In some embodiments, an IL-22polypeptide or IL-22 Fc fusion protein of the invention are used todelay development of a disease or to slow the progression of a disease.

In certain embodiments, a “subject in need thereof” in the context ofpreventing or treating a cardiovascular condition refers to a subjectdiagnosed with a cardiovascular disease or cardiovascular condition(CVD) or metabolic syndrome or exhibiting one or more conditionsassociated with CVD or metabolic syndrome, a subject who has beendiagnosed with or exhibited one or more conditions associated with CVDor metabolic syndrome in the past, or a subject who has been deemed atrisk of developing CVD or metabolic syndrome or one or more conditionsassociated with CVD or metabolic syndrome in the future due tohereditary or environmental factors. Therefore, in certain embodiments,a subject in need thereof can be a subject exhibiting a CVD or metabolicsyndrome or a condition associated with a CVD or metabolic syndrome or asubject that has exhibited a CVD or metabolic syndrome or a conditionassociated with a CVD or metabolic syndrome in the past or has beendeemed at risk for developing a CVD or metabolic syndrome or a conditionassociated with a CVD or metabolic syndrome in the future.

In treatment of a cardiovascular disease or condition, a therapeuticagent can directly alter the magnitude of response of a component of theimmune response, or render the disease more susceptible to treatment byother therapeutic agents, e.g., antibiotics, antifungals,anti-inflammatory agents, chemotherapeutics, etc. In treatment of anarterial disease, treatment might, for example, prevent or slow down theprogression of a disease. Thus, treatment of an arterial diseasespecifically includes the prevention, inhibition, or slowing down of thedevelopment of the condition, or of the progression from one stage ofthe condition to another, more advanced stage, or into a more severe,related condition.

The “pathology” of a disease or condition includes all phenomena thatcompromise the well-being of the subject. In the case of acardiovascular disease or condition, this includes, without limitation,atherosclerotic plaque formation (including stable orunstable/vulnerable plaques), atherosclerosis, arteriosclerosis,arteriolosclerosis, and elevated systemic lipopolysaccharide (LPS)exposure.

“Alleviation”, “alleviating” or equivalents thereof, refers to boththerapeutic treatment and prophylactic or preventative measures, whereinthe object is to ameliorate, prevent, slow down (lessen), decrease orinhibit a disease or condition, e.g., the formation of atheroscleroticplaques. Those in need of treatment include those already with thedisease or condition as well as those prone to having the disease orcondition or those in whom the disease or condition is to be prevented.

“Chronic” administration refers to administration of an agent(s) in acontinuous mode as opposed to an acute mode, so as to maintain theinitial therapeutic effect for an extended period of time.

“Intermittent” administration is treatment that is not consecutivelydone without interruption, but rather is cyclic in nature.

The term “package insert” is used to refer to instructions customarilyincluded in commercial packages of therapeutic products, that containinformation about the indications, usage, dosage, administration,combination therapy, contraindications and/or warnings concerning theuse of such therapeutic products.

“Percent (%) amino acid sequence identity” with respect to a referencepolypeptide sequence is defined as the percentage of amino acid residuesin a candidate sequence that are identical with the amino acid residuesin the reference polypeptide sequence, after aligning the sequences andintroducing gaps, if necessary, to achieve the maximum percent sequenceidentity, and not considering any conservative substitutions as part ofthe sequence identity. Alignment for purposes of determining percentamino acid sequence identity can be achieved in various ways that arewithin the skill in the art, for instance, using publicly availablecomputer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR)software. Those skilled in the art can determine appropriate parametersfor aligning sequences, including any algorithms needed to achievemaximal alignment over the full length of the sequences being compared.For purposes herein, however, % amino acid sequence identity values aregenerated using the sequence comparison computer program ALIGN-2. TheALIGN-2 sequence comparison computer program was authored by Genentech,Inc., and the source code has been filed with user documentation in theU.S. Copyright Office, Washington D.C., 20559, where it is registeredunder U.S. Copyright Registration No. TXU510087. The ALIGN-2 program ispublicly available from Genentech, Inc., South San Francisco, Calif., ormay be compiled from the source code. The ALIGN-2 program should becompiled for use on a UNIX operating system, including digital UNIXV4.0D. All sequence comparison parameters are set by the ALIGN-2 programand do not vary.

In situations where ALIGN-2 is employed for amino acid sequencecomparisons, the % amino acid sequence identity of a given amino acidsequence A to, with, or against a given amino acid sequence B (which canalternatively be phrased as a given amino acid sequence A that has orcomprises a certain % amino acid sequence identity to, with, or againsta given amino acid sequence B) is calculated as follows:100 times the fraction X/Ywhere X is the number of amino acid residues scored as identical matchesby the sequence alignment program ALIGN-2 in that program's alignment ofA and B, and where Y is the total number of amino acid residues in B. Itwill be appreciated that where the length of amino acid sequence A isnot equal to the length of amino acid sequence B, the % amino acidsequence identity of A to B will not equal the % amino acid sequenceidentity of B to A. Unless specifically stated otherwise, all % aminoacid sequence identity values used herein are obtained as described inthe immediately preceding paragraph using the ALIGN-2 computer program.As further examples of % amino acid sequence identity calculations usingthis method, below demonstrate how to calculate the % amino acidsequence identity of the amino acid sequence designated “ComparisonProtein” or “Reference Protein” to the amino acid sequence designated“IL-22”, wherein “IL-22” represents the amino acid sequence of an IL-22polypeptide of interest, “Comparison Protein” represents the amino acidsequence of a polypeptide against which the “IL-22” polypeptide ofinterest is being compared, and “X, “Y” and “Z” each represent differentamino acid residues.

As examples of % amino acid sequence identity calculations using thismethod, Tables 1 and 2 demonstrate how to calculate the % amino acidsequence identity of the amino acid sequence designated “ComparisonProtein” to the amino acid sequence designated “IL-22”, wherein “IL-22”represents the amino acid sequence of an IL-22 polypeptide of interest,“Comparison Protein” represents the amino acid sequence of a polypeptideagainst which the “IL-22” polypeptide of interest is being compared, and“X, “Y” and “Z” each represent different amino acid residues.

IL-22 XXXXXXXXXXXXXXX (Length = 15 amino acids) Reference XXXXXYYYYYYY(Length = 12 amino Protein acids)% amino acid sequence identity=(the number of identically matching aminoacid residues between the two polypeptide sequences) divided by (thetotal number of an acid residues of the IL-22 polypeptide)=5 divided by15=33.3%.

IL-22 XXXXXXXXXX (Length = 10 amino acids) Reference XXXXXYYYYYYZZYX(Length = 15 amino Protein acids)amino acid sequence identity=(the number of identically matching aminoacid residues between the two polypeptide sequences) divided by (thetotal number of amino acid residues of the IL-22 polypeptide)=5 dividedby 10=50%

“Stringency” of hybridization reactions is readily determinable by oneof ordinary skill in the art, and generally is an empirical calculationdependent upon probe length, washing temperature, and saltconcentration. In general, longer probes require higher temperatures forproper annealing, while shorter probes need lower temperatures.Hybridization generally depends on the ability of denatured DNA tore-anneal when complementary strands are present in an environment belowtheir melting temperature. The higher the degree of desired homologybetween the probe and hybridizable sequence, the higher the relativetemperature which can be used. As a result, it follows that higherrelative temperatures would tend to make the reaction conditions morestringent, while lower temperatures less so. For additional details andexplanation of stringency of hybridization reactions, see Ausubel etal., Current Protocols in Molecular Biology, Wiley IntersciencePublishers, (1995).

“Stringent conditions” or “high stringency conditions”, as definedherein, can be identified by those that: (1) employ low ionic strengthand high temperature for washing, for example 0.015 M sodiumchloride/0.0015 M sodium citrate/0.1% sodium dodecyl sulfate at 5OC; (2)employ during hybridization a denaturing agent, such as formamide, forexample, 50% (v/v) formamide with 0.1% bovine serum albumin/0.1%Ficoll/0.1% polyvinylpyrrolidone/50 mM sodium phosphate buffer at pH 6.5with 750 mM sodium chloride, 75 mM sodium citrate at 42° C.; or (3)overnight hybridization in a solution that employs 50% formamide, 5×SSC(0.75 M NaCl, 0.075 M sodium citrate), 50 mM sodium phosphate (pH 6.8),0.1% sodium pyrophosphate, 5×Denhardt's solution, sonicated salmon spermDNA (50 μg/ml), 0.1% SDS, and 10% dextran sulfate at 42° C., with a 10minute wash at 42° C. in 0.2×SSC (sodium chloride/sodium citrate)followed by a 10 minute high-stringency wash consisting of 0.1×SSCcontaining EDTA at 55° C.

“Moderately stringent conditions” can be identified as described bySambrook et al., Molecular Cloning: A Laboratory Manual. New York: ColdSpring Harbor Press, 1989, and include the use of washing solution andhybridization conditions (e.g., temperature, ionic strength, and % SDS)less stringent that those described above. An example of moderatelystringent conditions is overnight incubation at 37° C. in a solutioncomprising: 20% formamide, 5×SSC (150 mM NaCl, 15 mM trisodium citrate),50 mM sodium phosphate (pH 7.6), 5×Denhardt's solution, 10% dextransulfate, and 20 mg/ml denatured sheared salmon sperm DNA, followed bywashing the filters in 1×SSC at about 37-500 C. The skilled artisan willrecognize how to adjust the temperature, ionic strength, etc. asnecessary to accommodate factors such as probe length and the like.

The term “agonist” is used in the broadest sense and includes anymolecule that partially or fully mimics a biological activity of anIL-22 polypeptide. Also encompassed by “agonist” are molecules thatstimulate the transcription or translation of mRNA encoding thepolypeptide.

Suitable agonist molecules include, e.g., agonist antibodies or antibodyfragments; a native polypeptide; fragments or amino acid sequencevariants of a native polypeptide; peptides; antisense oligonucleotides;small organic molecules; and nucleic acids that encode polypeptidesagonists or antibodies. Reference to “an” agonist encompasses a singleagonist or a combination of two or more different agonists.

The term “IL-22 agonist” is used in the broadest sense, and includes anymolecule that mimics a qualitative biological activity (as hereinabovedefined) of a native sequence IL-22 polypeptide. IL-22 agonistsspecifically include IL-22-Fc or IL-22 Ig polypeptides (immunoadhesins),but also small molecules mimicking at least one IL-22 biologicalactivity. Preferably, the biological activity is binding of the IL-22receptor, interacting with IL-22BP, facilitating an innate immuneresponse pathway, or in the case of a cardiovascular disease orcondition, to affect the formation of atherosclerotic plaques, inparticular to inhibit formation of atherosclerotic plaque formation.Inhibition of plaque formation can be assessed by any suitable imagingmethod known to those of ordinary skill in the art.

IL-22R1 pairs with other proteins to form heterodimers as the receptorsfor certain IL-10 family members. See Quyang et al., 2011, supra. Thus,in certain embodiments, IL-22 agonists may include an IL-22 receptoragonist, including a cytokine (or a fusion protein or agonist thereof)that binds to and triggers downstream signaling of the IL-22 R1. Incertain embodiments, the IL-22 agonists include an IL-22R1 agonist,including without limitation an anti-IL-22R1 agonist antibody; an IL-20agonist, including without limitation IL-20 polypeptide or IL-20 Fcfusion protein; and an IL-24 agonist, including without limitation IL-24polypeptide or IL-24 fusion protein. In certain other embodiments, theIL-22R1 agonists include an IL-19 agonist, including without limitationIL-19 polypeptide or IL-19 Fc fusion protein; and an IL-26 agonist,including without limitation IL-26 polypeptide or IL-26 Fc fusionprotein. Exemplary sequences for IL-19 (GenBank Accession No.AAG16755.1, SEQ ID NO:77), IL-20 (GenBank Accession No. AAH69311.1, SEQID NO:78), IL-24 (GenBank Accession No. AAH09681.1, SEQ ID NO:79) andIL-26 (GenBank Accession No. NP_060872.1, SEQ ID NO:80) are providedherein. In certain embodiments, an IL-19 polypeptide comprises the aminoacid sequence of SEQ ID NO:77 or the mature protein without the signalpeptide. In certain other embodiments, an IL-20 polypeptide comprisesthe amino acid sequence of SEQ ID NO:78 or the mature protein withoutthe signal peptide. In yet other embodiments, an IL-24 polypeptidecomprises the amino acid sequence of SEQ ID NO:79 or the mature proteinwithout the signal peptide. In certain other embodiments, an IL-26polypeptide comprises the amino acid sequence of SEQ ID NO:80 or themature protein without the signal peptide.

A “small molecule” is defined herein to have a molecular weight belowabout 600, preferably below about 1000 daltons.

An “agonist antibody,” as used herein, is an antibody which partially orfully mimics a biological activity of an IL-22 polypeptide.

The term “pharmaceutical formulation” or “pharmaceutical composition”refers to a preparation which is in such form as to permit thebiological activity of an active ingredient contained therein to beeffective, and which contains no additional components which areunacceptably toxic to a subject to which the formulation would beadministered.

A “pharmaceutically acceptable carrier” refers to an ingredient in apharmaceutical formulation, other than an active ingredient, which isnontoxic to a subject. A pharmaceutically acceptable carrier includes,but is not limited to, a buffer, excipient, diluent, stabilizer, orpreservative.

The term “variable region” or “variable domain” refers to the domain ofan antibody heavy or light chain that is involved in binding theantibody to antigen. The variable domains of the heavy chain and lightchain (VH and VL, respectively) of a native antibody generally havesimilar structures, with each domain comprising four conserved frameworkregions (FRs) and three hypervariable regions (HVRs). (See, e.g., Kindtet al. Kuby Immunology, 6^(th) ed., W. H. Freeman and Co., page 91(2007).) A single VH or VL domain may be sufficient to conferantigen-binding specificity. Furthermore, antibodies that bind aparticular antigen may be isolated using a VH or VL domain from anantibody that binds the antigen to screen a library of complementary VLor VH domains, respectively. See, e.g., Portolano et al., J. Immunol.150:880-887 (1993); Clarkson et al., Nature 352:624-628 (1991).

The term “vector,” as used herein, refers to a nucleic acid moleculecapable of propagating another nucleic acid to which it is linked. Theterm includes the vector as a self-replicating nucleic acid structure aswell as the vector incorporated into the genome of a host cell intowhich it has been introduced. Certain vectors are capable of directingthe expression of nucleic acids to which they are operatively linked.Such vectors are referred to herein as “expression vectors.”

II. Compositions and Methods

In one aspect, the invention is based, in part, on compositionscomprising therapeutics that ameliorate IL-22 associated diseases ordisorders by increasing IL-22 activities or signaling. In certainembodiments, IL-22 polypeptide and IL-22 Fc fusion proteins that bind toand activate IL-22 receptor are provided. IL-22 Fc fusion proteins ofthe invention are useful, e.g., for the diagnosis or treatment of IL-22associated diseases such as inflammatory bowel disease and acceleratingwound healing. In addition, IL-22 polypeptide and IL-22 Fc fusionproteins for the treatment of other IL-22 associated diseases forexample cardiovascular conditions, metabolic syndrome and acceleratingdiabetic wound healing are also provided.

A. Exemplary IL-22 Polypeptide

IL-22 polypeptide as used herein includes a polypeptide comprising anamino acid sequence comprising SEQ ID NO:71 (human IL-22 with theendogenous IL-22 leader sequence) (see FIG. 31), or a polypeptidecomprising an amino acid sequence that has at least 95% sequenceidentity with SEQ ID NO:71. In certain embodiments, the IL-22polypeptide comprises an amino acid sequence comprising SEQ ID NO:4(human IL-22 without a leader sequence) or a polypeptide comprising anamino acid sequence that has at least 95% sequence identity. In certainembodiments, the IL-22 polypeptide comprises an amino acid sequencecomprising SEQ ID NO:4. In certain embodiments, the IL-22 polypeptidedoes not comprise an Fc fusion. The preparation of native IL-22molecules, along with their nucleic acid and polypeptide sequences, canbe achieved through methods known to those of ordinary skill in the art.For example, IL-22 polypeptides can be produced by culturing cellstransformed or transfected with a vector containing IL-22 nucleic acid.It is, of course, contemplated that alternative methods, which are wellknown in the art, can be employed to prepare IL-22. For instance, theIL-22 sequence, or portions thereof, can be produced by direct peptidesynthesis using solid-phase techniques (see, e.g., Stewart et al., 1969,Solid-Phase Peptide Synthesis, W.H. Freeman Co., San Francisco, Calif.(1969); Merrifield, J. Am. Chem. Soc., 1963, 85:2149-2154). In vitroprotein synthesis can be performed using manual techniques or byautomation. Automated synthesis can be accomplished, for instance, usingan Applied Biosystems Peptide Synthesizer (Foster City, Calif.) usingmanufacturer's instructions. Various portions of IL-22 can be chemicallysynthesized separately and combined using chemical or enzymatic methodsto produce the full-length IL-22.

IL-22 variants can be prepared by introducing appropriate nucleotidechanges into the DNA encoding a native sequence IL-22 polypeptide, or bysynthesis of the desired IL-22 polypeptide. Those skilled in the artwill appreciate that amino acid changes can alter post-translationalprocesses of IL-22, such as changing the number or position ofglycosylation sites or altering the membrane anchoring characteristics.

Variations in the native sequence IL-22 polypeptides described hereincan be made, for example, using any of the techniques and guidelines forconservative and non-conservative mutations set forth, for instance, inU.S. Pat. No. 5,364,934. Variations can be a substitution, deletion orinsertion of one or more codons encoding a native sequence or variantIL-22 that results in a change in its amino acid sequence as comparedwith a corresponding native sequence or variant IL-22. Optionally thevariation is by substitution of at least one amino acid with any otheramino acid in one or more of the domains of a native sequence IL-22polypeptide.

Guidance in determining which amino acid residue can be inserted,substituted or deleted without adversely affecting the desired activitycan be found by comparing the sequence of the IL-22 with that ofhomologous known protein molecules and minimizing the number of aminoacid sequence changes made in regions of high homology. Amino acidsubstitutions can be the result of replacing one amino acid with anotheramino acid having similar structural and/or chemical properties, such asthe replacement of a leucine with a serine, i.e., conservative aminoacid replacements. Insertions or deletions can optionally be in therange of 1 to 5 amino acids. The variation allowed can be determined bysystematically making insertions, deletions or substitutions of aminoacids in the sequence and testing the resulting variants for activity inthe in vitro assay described in the Examples below.

In particular embodiments, conservative substitutions of interest areshown in Table 1 under the heading of preferred substitutions. If suchsubstitutions result in a change in biological activity, then moresubstantial changes, denominated exemplary substitutions in Table 1, oras further described below in reference to amino acid classes, areintroduced and the products screened.

The variations can be made using methods known in the art such asoligonucleotide-mediated (site-directed) mutagenesis, alanine scanning,and PCR mutagenesis. Site-directed mutagenesis (Carter et al., 1986,Nucl. Acids Res, 13:4331; Zoller et al., 1987, Nucl. Acids Res.,10:6487), cassette mutagenesis (Wells et al., 1985, Gene, 34:315),restriction selection mutagenesis (Wells et al., 1986, Philos. Trans. R.Soc. London SerA, 317:415) or other known techniques can be performed onthe cloned DNA to produce the IL-22 variant DNA.

Fragments of an IL-22 polypeptide of the present invention are alsoprovided herein. Such fragments can be truncated at the N-terminus orC-terminus, or can lack internal residues, for example, when comparedwith a full length native protein. Certain fragments lack amino acidresidues that are not essential for a desired biological activity of anIL-22 polypeptide of the present invention. Accordingly, in certainembodiments, a fragment of an IL-22 polypeptide is biologically active.In certain embodiments, a fragment of full length IL-22 lacks theN-terminal signal peptide sequence.

Covalent modifications of native sequence and variant IL-22 polypeptidesare included within the scope of this invention. One type of covalentmodification includes reacting targeted amino acid residues of IL-22with an organic derivatizing agent that is capable of reacting withselected side chains or the N- or C-terminal residues of the IL-22polypeptide. Derivatization with bifunctional agents is useful, forinstance, for crosslinking IL-22 to a water-insoluble support matrix orsurface, for example, for use in the method for purifying anti-IL-22antibodies. Commonly used crosslinking agents include, e.g.,1,1-bis(diazo-acetyl)-2-phenylethane, glutaraldehyde,N-hydroxysuccinimide esters, for example, esters with 4-azidosalicylicacid, homobifunctional imidoesters, including disuccinimidyl esters suchas 3,3′-dithiobis(succinimidyl-propionate), bifunctional maleimides suchas bis-N-maleimido-1,8-octane and agents such asmethyl-3-[(p-azidophenyl)dithio]propioimidate.

Other modifications include deamidation of glutaminyl and asparaginylresidues to the corresponding glutamyl and aspartyl residues,respectively, hydroxylation of proline and lysine, phosphorylation ofhydroxyl groups of seryl or threonyl residues, methylation of the.alpha.-amino groups of lysine, arginine, and histidine side chains (T.E. Creighton, 1983, Proteins: Structure and Molecular Properties, W. H.Freeman & Co., San Francisco, pp. 79-86i), acetylation of the N-terminalamine, and amidation of any C-terminal carboxyl group.

Another type of covalent modification of the IL-22 polypeptides includedwithin the scope of this invention comprises altering the nativeglycosylation pattern of the polypeptides. “Altering the nativeglycosylation pattern” is intended for purposes herein to mean deletingone or more carbohydrate moieties found in native sequence IL-22, and/oradding one or more glycosylation sites that are not present in thenative sequence IL-22, and/or alteration of the ratio and/or compositionof the sugar residues attached to the glycosylation site(s).

Glycosylation of polypeptides is typically either N-linked or O-linked.N-linked glycosylation refers to the attachment of the carbohydratemoiety to the side-chain of an asparagine residue. The tripeptidesequences, asparagine-X-serine and asparagine-X-threonine, wherein X isany amino acid except proline, are recognition sequences for enzymaticattachment of the carbohydrate moiety to the asparagine side chain.O-linked glycosylation refers to the attachment of one of the sugarsN-acetylgalactosamine, galactose, or xylose to a hydroxyamino acid, mostcommonly serine or threonine, although 5-hydroxyproline or5-hydroxylysine can also be involved in O-linked glycosylation. Additionof glycosylation sites to the IL-22 polypeptide can be accomplished byaltering the amino acid sequence. The alteration can be made, forexample, by the addition of, or substitution by, one or more serine orthreonine residues to the native sequence IL-22 (for N-linkedglycosylation sites), or the addition of a recognition sequence forO-linked glycosylation. The IL-22 amino acid sequence can optionally bealtered through changes at the DNA level, particularly by mutating theDNA encoding the IL-22 polypeptide at preselected bases such that codonsare generated that will translate into the desired amino acids.

Another means of increasing the number of carbohydrate moieties on theIL-22 polypeptide is by chemical or enzymatic coupling of glycosides tothe polypeptide. Such methods are described in the art, e.g., in WO87/05330 published 11 Sep. 1987, and in Aplin and Wriston, CRC Crit.Rev. Biochem., pp. 259-306 (1981).

Removal of carbohydrate moieties present on an IL-22 polypeptide can beaccomplished chemically or enzymatically or by mutational substitutionof codons encoding for amino acid residues that serve as targets forglycosylation. Chemical deglycosylation techniques are known in the artand described, for instance, by Hakimuddin, et al., Arch. Biochem.Biophys., 259:52 (1987) and by Edge et al., Anal. Biochem., 118:131(1981). Enzymatic cleavage of carbohydrate moieties on polypeptides canbe achieved by the use of a variety of endo- and exo-glycosidases asdescribed by Thotakura et al., Meth. Enzymol., 138:350 (1987).

Another type of covalent modification of IL-22 comprises linking theIL-22 polypeptide to one of a variety of nonproteinaceous polymers,e.g., polyethylene glycol, polypropylene glycol, or polyoxyalkylenes,for example in the manner set forth in U.S. Pat. Nos. 4,640,835;4,496,689; 4,301,144; 4,670,417; 4,791,192 or 4,179,337. The nativesequence and variant IL-22 can also be modified in a way to form achimeric molecule comprising IL-22, including fragments of IL-22, fusedto another, heterologous polypeptide or amino acid sequence.

In one embodiment, such a chimeric molecule comprises a fusion of IL-22with a tag polypeptide which provides an epitope to which an anti-tagantibody can selectively bind. The epitope tag is generally placed atthe amino- or carboxyl-terminus of the IL-22 polypeptide. The presenceof such epitope-tagged forms of the IL-22 polypeptide can be detectedusing an antibody against the tag polypeptide. Also, provision of theepitope tag enables the IL-22 polypeptide to be readily purified byaffinity purification using an anti-tag antibody or another type ofaffinity matrix that binds to the epitope tag. Various tag polypeptidesand their respective antibodies are well known in the art. Examplesinclude poly-histidine (poly-his) or poly-histidine-glycine(poly-his-gly) tags; the flu HA tag polypeptide and its antibody 12CA5(Field et al., 1988, Mol. Cell. Biol., 8:2159-2165); the c-myc tag andthe 8F9, 3C7, 6E10, G4, and 9E10 antibodies thereto (Evan et al., 1985,Molecular and Cellular Biology, 5:3610-3616); and the Herpes Simplexvirus glycoprotein D (gD) tag and its antibody (Paborsky et al., 1990,Protein Engineering, 3(6):547-553). Other tag polypeptides include theFlag-peptide (Hopp et al., 1988, BioTechnology, 6:1204-1210); the KT3epitope peptide (Martin et al., 1992, Science, 255:192-194); an.quadrature.-tubulin epitope peptide (Skinner et al., 1991, J. Biol.Chem., 266:15163-15166); and the T7 gene 10 protein peptide tag(Lutz-Freyermuth et al., 1990, Proc. Natl. Acad. Sci. USA,87:6393-6397).

In another embodiment, the chimeric molecule can comprise a fusion ofthe IL-22 polypeptide or a fragment thereof with an immunoglobulin or aparticular region of an immunoglobulin. For a bivalent form of thechimeric molecule, such a fusion can be to the Fc region of an IgGmolecule. These fusion polypeptides are antibody-like molecules whichcombine the binding specificity of a heterologous protein (an “adhesin”)with the effector functions of immunoglobulin constant domains, and areoften referred to as immunoadhesins. Structurally, the immunoadhesinscomprise a fusion of an amino acid sequence of IL-22, or a variantthereof, and an immunoglobulin constant domain sequence. The adhesinpart of an immunoadhesin molecule typically is a contiguous amino acidsequence comprising at least the binding site of a receptor or a ligand.The immunoglobulin constant domain sequence in the immunoadhesin can beobtained from any immunoglobulin, such as IgG1, IgG2, IgG3, or IgG4subtypes, IgA (including IgA1 and IgA2), IgE, IgD or IgM. In certainembodiments, the IL-22 Fc fusion protein exhibits modified effectoractivities.

The IL-22 polypeptide, or a fragment thereof, can be fused, for example,to an immunoglobulin heavy chain constant region sequence to produce anIL-22-Ig fusion protein (e.g., IL-22 Fc fusion protein). The IL-22polypeptide can be human or murine IL-22. The immunoglobulin heavy chainconstant region sequence can be human or murine immunoglobulin heavychain constant region sequence.

B. Exemplary IL-22 Fc Fusion Protein

In one aspect, the invention provides isolated IL-22 fusion protein. Incertain embodiments, the IL-22 fusion protein binds to and induces IL-22receptor activity or signaling and/or is an agonist of IL-22 receptoractivity.

In another aspect, an IL-22 Fc fusion protein comprises a polypeptidehaving at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or100% sequence identity to the amino acid sequence of SEQ ID NO:4. Inother embodiments, the IL-22 Fc fusion protein comprises a polypeptidehaving at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%sequence identity contains substitutions (e.g., conservativesubstitutions), insertions, or deletions relative to the referencesequence, but an IL-22 Fc fusion protein comprising that sequenceretains the ability to bind to IL-22 receptor. In certain embodiments, atotal of 1 to 10 amino acids have been substituted, inserted and/ordeleted in SEQ ID NOs:8, 10, 12, 14, 24 or 26. In certain embodiments,substitutions, insertions, or deletions occur in regions outside theIL22 (i.e., in the Fc). In certain particular embodiments, theC-terminus Lys residue of Fc is deleted. In certain other embodiments,the C-terminus Gly and Lys residues of Fc are both deleted.

In certain embodiments, IL-22 Fc fusion proteins variants having one ormore amino acid substitutions are provided. Conservative substitutionsare shown in Table 1 under the heading of “preferred substitutions.”More substantial changes are provided in Table 1 under the heading of“exemplary substitutions,” and as further described below in referenceto amino acid side chain classes. Amino acid substitutions may beintroduced into the IL-22 Fc fusion protein and the products screenedfor a desired activity, e.g., retained/improved IL-22 receptor binding,decreased immunogenicity, or improved IL-22 receptor signaling.

TABLE 1 Original Exemplary Preferred Residue Substitutions SubstitutionsAla (A) Val; Leu; Ile Val Arg (R) Lys; Gln; Asn Lys Asn (N) Gln; His;Asp, Lys; Arg Gln Asp (D) Glu; Asn Glu Cys (C) Ser; Ala Ser Gln (Q) Asn;Glu Asn Glu (E) Asp; Gln Asp Gly (G) Ala Ala His (H) Asn; Gln; Lys; ArgArg Ile (I) Leu; Val; Met; Ala; Phe; Norleucine Leu Leu (L) Norleucine;Ile; Val; Met; Ala; Phe Ile Lys (K) Arg; Gln; Asn Arg Met (M) Leu; Phe;Ile Leu Phe (F) Trp; Leu; Val; Ile; Ala; Tyr Tyr Pro (P) Ala Ala Ser (S)Thr Thr Thr (T) Val; Ser Ser Trp (W) Tyr; Phe Tyr Tyr (Y) Trp; Phe; Thr;Ser Phe Val (V) Ile; Leu; Met; Phe; Ala; Norleucine LeuAmino acids may be grouped according to common side-chain properties:

(1) hydrophobic: Norleucine, Met, Ala, Val, Leu, Ile;

(2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gln;

(3) acidic: Asp, Glu;

(4) basic: His, Lys, Arg;

(5) residues that influence chain orientation: Gly, Pro;

(6) aromatic: Trp, Tyr, Phe.

Non-conservative substitutions will entail exchanging a member of one ofthese classes for another class.

A useful method for identification of residues or regions of a proteinthat may be targeted for mutagenesis is called “alanine scanningmutagenesis” as described by Cunningham and Wells (1989) Science,244:1081-1085. In this method, a residue or group of target residues(e.g., charged residues such as arg, asp, his, lys, and glu) areidentified and replaced by a neutral or negatively charged amino acid(e.g., alanine or polyalanine) to determine whether the interaction ofthe protein with its binding partner is affected. Further substitutionsmay be introduced at the amino acid locations demonstrating functionalsensitivity to the initial substitutions. Alternatively, oradditionally, a crystal structure of a protein complex (e.g., acytokine-receptor complex) can be used to identify contact pointsbetween a protein and its binding partner. Such contact residues andneighboring residues may be targeted or eliminated as candidates forsubstitution. Variants may be screened to determine whether they containthe desired properties.

Amino acid sequence insertions include amino- and/or carboxyl-terminalfusions ranging in length from one residue to polypeptides containing ahundred or more residues, as well as intrasequence insertions of singleor multiple amino acid residues.

a) Glycosylation Variants

In certain embodiments, an Fc fusion protein provided herein is alteredto increase or decrease the extent to which the fusion protein,especially the Fc portion of the fusion protein, is glycosylated.Addition or deletion of glycosylation sites to a protein may beconveniently accomplished by altering the amino acid sequence such thatone or more glycosylation sites is created or removed.

Where the fusion protein comprises an Fc region, the carbohydrateattached thereto may be altered. Native antibodies produced by mammaliancells typically comprise a branched, biantennary oligosaccharide that isgenerally attached by an N-linkage to Asn297 of the CH2 domain of the Fcregion. See, e.g., Wright et al. TIBTECH 15:26-32 (1997). Theoligosaccharide may include various carbohydrates, e.g., mannose,N-acetyl glucosamine (GlcNAc), galactose, and sialic acid, as well as afucose attached to a GlcNAc in the “stem” of the biantennaryoligosaccharide structure. In some embodiments, modifications of theoligosaccharide in an antibody or the Fc region of an antibody may bemade in order to create Fc variants with certain improved properties.

The amount of fucose attached to the CH2 domain of the Fc region can bedetermined by calculating the average amount of fucose within the sugarchain at Asn297, relative to the sum of all glycostructures attached toAsn 297 or N297 (e. g. complex, hybrid and high mannose structures) asmeasured by MALDI-TOF mass spectrometry, as described in WO 2008/077546,for example. Asn297 refers to the asparagine residue located at aboutposition 297 in the Fc region (EU numbering of Fc region residues);however, Asn297 may also be located about ±3 amino acids upstream ordownstream of position 297, i.e., between positions 294 and 300, due tominor sequence variations in antibodies. Such fucosylation variants mayhave improved ADCC function. See, e.g., US Patent Publication Nos. US2003/0157108 (Presta, L.); US 2004/0093621 (Kyowa Hakko Kogyo Co., Ltd).Examples of publications related to “defucosylated” or“fucose-deficient” antibody variants include: US 2003/0157108; WO2000/61739; WO 2001/29246; US 2003/0115614; US 2002/0164328; US2004/0093621; US 2004/0132140; US 2004/0110704; US 2004/0110282; US2004/0109865; WO 2003/085119; WO 2003/084570; WO 2005/035586; WO2005/035778; WO2005/053742; WO2002/031140; Okazaki et al. J. Mol. Biol.336:1239-1249 (2004); Yamane-Ohnuki et al. Biotech. Bioeng. 87: 614(2004). Examples of cell lines capable of producing defucosylatedantibodies include Lec13 CHO cells deficient in protein fucosylation(Ripka et al. Arch. Biochem. Biophys. 249:533-545 (1986); US Pat Appl NoUS 2003/0157108 A1, Presta, L; and WO 2004/056312 A1, Adams et al.,especially at Example 11), and knockout cell lines, such asalpha-1,6-fucosyltransferase gene, FUT8, knockout CHO cells (see, e.g.,Yamane-Ohnuki et al. Biotech. Bioeng. 87: 614 (2004); Kanda, Y. et al.,Biotechnol. Bioeng., 94(4):680-688 (2006); and WO2003/085107).

Antibodies variants are further provided with bisected oligosaccharides,e.g., in which a biantennary oligosaccharide attached to the Fc regionof the antibody is bisected by GlcNAc. Such antibody variants may havereduced fucosylation and/or improved ADCC function. Examples of suchantibody variants are described, e.g., in WO 2003/011878 (Jean-Mairet etal.); U.S. Pat. No. 6,602,684 (Umana et al.); and US 2005/0123546 (Umanaet al.). Antibody variants with at least one galactose residue in theoligosaccharide attached to the Fc region are also provided. Suchantibody variants may have improved CDC function. Such antibody variantsare described, e.g., in WO 1997/30087 (Patel et al.); WO 1998/58964(Raju, S.); and WO 1999/22764 (Raju, S.).

b) Fc Region Variants

In certain embodiments, one or more amino acid modifications may beintroduced into the Fc region of an Fc fusion protein provided herein,thereby generating an Fc region variant. The Fc region variant maycomprise a human Fc region sequence (e.g., a human IgG1, IgG2, IgG3 orIgG4 Fc region) comprising an amino acid modification (e.g. asubstitution) at one or more amino acid positions.

In certain embodiments, the invention contemplates an Fc variant thatpossesses some but not all effector functions, which make it a desirablecandidate for applications in which the half life of the antibody or afusion protein comprising an Fc region in vivo is important yet certaineffector functions (such as complement and ADCC) are unnecessary ordeleterious. In vitro and/or in vivo cytotoxicity assays can beconducted to confirm the reduction/depletion of CDC and/or ADCCactivities. For example, Fc receptor (FcR) binding assays can beconducted to ensure that the antibody or Fc lacks FcγR binding (hencelikely lacking ADCC activity), but retains FcRn binding ability. Theprimary cells for mediating ADCC, NK cells, express FcγRIII only,whereas monocytes express FcγRI, FcγRII and FcγRIII. FcR expression onhematopoietic cells is summarized in Table 3 on page 464 of Ravetch andKinet, Annu. Rev. Immunol. 9:457-492 (1991). Non-limiting examples of invitro assays to assess ADCC activity of a molecule of interest isdescribed in U.S. Pat. No. 5,500,362 (see, e.g. Hellstrom, I. et al.Proc. Nat'l Acad. Sci. USA 83:7059-7063 (1986)) and Hellstrom, I et al.,Proc. Nat'l Acad. Sci. USA 82:1499-1502 (1985); U.S. Pat. No. 5,821,337(see Bruggemann, M. et al., J. Exp. Med. 166:1351-1361 (1987)).Alternatively, non-radioactive assays methods may be employed (see, forexample, ACTI™ non-radioactive cytotoxicity assay for flow cytometry(CellTechnology, Inc. Mountain View, Calif.; and CytoTox 96®non-radioactive cytotoxicity assay (Promega, Madison, Wis.). Usefuleffector cells for such assays include peripheral blood mononuclearcells (PBMC) and Natural Killer (NK) cells. Alternatively, oradditionally, ADCC activity of the molecule of interest may be assessedin vivo, e.g., in an animal model such as that disclosed in Clynes etal. Proc. Nat'l Acad. Sci. USA 95:652-656 (1998). C1q binding assays mayalso be carried out to confirm that the antibody or Fc is unable to bindC1q and hence lacks CDC activity. See, e.g., C1q and C3c binding ELISAin WO 2006/029879 and WO 2005/100402. To assess complement activation, aCDC assay may be performed (see, for example, Gazzano-Santoro et al., J.Immunol. Methods 202:163 (1996); Cragg, M. S. et al., Blood101:1045-1052 (2003); and Cragg, M. S. and M. J. Glennie, Blood103:2738-2743 (2004)). FcRn binding and in vivo clearance/half-lifedeterminations can also be performed using methods known in the art(see, e.g., Petkova, S. B. et al., Int'l. Immunol. 18(12):1759-1769(2006)).

Antibodies with reduced effector function include those withsubstitution of one or more of Fc region residues 238, 265, 269, 270,297, 327 and 329 (U.S. Pat. No. 6,737,056). Such Fc mutants include Fcmutants with substitutions at two or more of amino acid positions 265,269, 270, 297 and 327, including the so-called “DANA” Fc mutant withsubstitution of residues 265 and 297 to alanine (U.S. Pat. No.7,332,581).

Certain antibody or Fc variants with improved or diminished binding toFcRs are described. (See, e.g., U.S. Pat. No. 6,737,056; WO 2004/056312,and Shields et al., J. Biol. Chem. 9(2): 6591-6604 (2001).)

In certain embodiments, an IL-22 Fc fusion protein comprises an Fcvariant with one or more amino acid substitutions which reduce ADCC,e.g., substitution at position 297 of the Fc region to remove theN-glycosylation site and yet retain FcRn binding activity (EU numberingof residues).

In some embodiments, alterations are made in the Fc region that resultin diminished C1q binding and/or Complement Dependent Cytotoxicity(CDC), e.g., as described in U.S. Pat. No. 6,194,551, WO 99/51642, andIdusogie et al. J. Immunol. 164: 4178-4184 (2000).

Antibodies with increased half lives and improved binding to theneonatal Fc receptor (FcRn), which is responsible for the transfer ofmaternal IgGs to the fetus (Guyer et al., J. Immunol. 117:587 (1976) andKim et al., J. Immunol. 24:249 (1994)), are described inUS2005/0014934A1 (Hinton et al.). Those antibodies comprise an Fc regionwith one or more substitutions therein which improve binding of the Fcregion to FcRn. Such Fc variants include those with substitutions at oneor more of Fc region residues: 238, 256, 265, 272, 286, 303, 305, 307,311, 312, 317, 340, 356, 360, 362, 376, 378, 380, 382, 413, 424 or 434,e.g., substitution of Fc region residue 434 (U.S. Pat. No. 7,371,826).

See also Duncan & Winter, Nature 322:738-40 (1988); U.S. Pat. Nos.5,648,260; 5,624,821; and WO 94/29351 concerning other examples of Fcregion variants.

c) Cysteine Engineered Variants

In certain embodiments, it may be desirable to create cysteineengineered Fc fusion protein, in which one or more residues of the Fcregion of an antibody are substituted with cysteine residues. Inparticular embodiments, the substituted residues occur at accessiblesites of the Fc. By substituting those residues with cysteine, reactivethiol groups are thereby positioned at accessible sites of the Fc andmay be used to conjugate the Fc to other moieties, such as drug moietiesor linker-drug moieties, to create an immunoconjugate, as describedfurther herein. For example, S400 (EU numbering) of the heavy chain Fcregion can be substituted with Cysteine. See e.g., U.S. Pat. No.7,521,541.

C. Recombinant Methods and Compositions

The IL-22 polypeptides can be prepared by routine recombinant methods,e.g., culturing cells transformed or transfected with a vectorcontaining a nucleic acid encoding an IL-22 polypeptide, a fragment orvariant thereof, or fusion protein comprising the same. Host cellscomprising any such vector are also provided. By way of example, hostcells can be CHO cells, E. coli, or yeast. A process for producing anyof the herein described polypeptides is further provided and comprisesculturing host cells under conditions suitable for expression of thedesired polypeptide and recovering the desired polypeptide from the cellculture.

Host cells are transfected or transformed with expression or cloningvectors described herein for IL-22 polypeptide production and culturedin conventional nutrient media modified as appropriate for inducingpromoters, selecting transformants, or amplifying the genes encoding thedesired sequences. The culture conditions, such as media, temperature,pH and the like, can be selected by the skilled artisan without undueexperimentation. In general, principles, protocols, and practicaltechniques for maximizing the productivity of cell cultures can be foundin Mammalian Cell Biotechnology: A Practical Approach, M. Butler, ed.(IRL Press, 1991) and Sambrook et al., supra.

Methods of transfection are known to the ordinarily skilled artisan, forexample, by CaPO₄ and electroporation. Depending on the host cell used,transformation is performed using standard techniques appropriate tosuch cells. The calcium treatment employing calcium chloride, asdescribed in Sambrook et al., supra, or electroporation is generallyused for prokaryotes or other cells that contain substantial cell-wallbarriers. Infection with Agrobacterium tumefaciens is used fortransformation of certain plant cells, as described by Shaw et al.,Gene, 23:315 (1983) and WO 89/05859 published 29 Jun. 1989. Formammalian cells without such cell walls, the calcium phosphateprecipitation method of Graham and van der Eb, Virology, 52:456-457(1978) can be employed. General aspects of mammalian cell host systemtransformations have been described in U.S. Pat. No. 4,399,216.Transformations into yeast are typically carried out according to themethod of Van Solingen et al., J. Bact, 130:946 (1977) and Hsiao et al.,Proc. Natl. Acad. Sci. (USA), 76:3829 (1979). However, other methods forintroducing DNA into cells, such as by nuclear microinjection,electroporation, bacterial protoplast fusion with intact cells, orpolycations, e.g., polybrene, polyornithine, can also be used. Forvarious techniques for transforming mammalian cells, see Keown et al.,Methods in Enzymology, 185:527-537 (1990) and Mansour et al., Nature,336:348-352 (1988).

Recombinantly expressed polypeptides of the present invention can berecovered from culture medium or from host cell lysates. The followingprocedures are exemplary of suitable purification procedures: byfractionation on an ion-exchange column; ethanol precipitation; reversephase HPLC; chromatography on silica or on a cation-exchange resin suchas DEAE; chromatofocusing; SDS-PAGE; ammonium sulfate precipitation; gelfiltration using, for example, Sephadex G-75; protein A Sepharosecolumns to remove contaminants such as IgG; and metal chelating columnsto bind epitope-tagged forms of a polypeptide of the present invention.Various methods of protein purification can be employed and such methodsare known in the art and described for example in Deutscher, Methods inEnzymology, 182 (1990); Scopes, Protein Purification: Principles andPractice, Springer-Verlag, New York (1982). The purification step(s)selected will depend, for example, on the nature of the productionprocess used and the particular polypeptide produced.

Alternative methods, which are well known in the art, can be employed toprepare a polypeptide of the present invention. For example, a sequenceencoding a polypeptide or portion thereof, can be produced by directpeptide synthesis using solid-phase techniques (see, e.g., Stewart etal., 1969, Solid-Phase Peptide Synthesis, W.H. Freeman Co., SanFrancisco, Calif.; Merrifield, J. 1963, Am. Chem. Soc., 85:2149-2154. Invitro protein synthesis can be performed using manual techniques or byautomation. Automated synthesis can be accomplished, for instance, usingan Applied Biosystems Peptide Synthesizer (Foster City, Calif.) usingmanufacturer's instructions. Various portions of a polypeptide of thepresent invention or portion thereof can be chemically synthesizedseparately and combined using chemical or enzymatic methods to producethe full-length polypeptide or portion thereof.

In other embodiments, the invention provides chimeric moleculescomprising any of the herein described polypeptides fused to aheterologous polypeptide or amino acid sequence. Examples of suchchimeric molecules include, but are not limited to, any of the hereindescribed polypeptides fused to an epitope tag sequence or an Fc regionof an immunoglobulin.

Suitable host cells for cloning or expressing the DNA in the vectorsherein include prokaryote, yeast, or higher eukaryote cells. Suitableprokaryotes include but are not limited to eubacteria, such asGram-negative or Gram-positive organisms, for example,Enterobacteriaceae such as E. coli. Various E. coli strains are publiclyavailable, such as E. coli K12 strain MM294 (ATCC 31,446); E. coli X1776(ATCC 31,537); E. coli strain W3110 (ATCC 27,325) and K5 772 (ATCC53,635).

In addition to prokaryotes, eukaryotic microbes such as filamentousfungi or yeast are suitable cloning or expression hosts forIL-22-encoding vectors. Saccharomyces cerevisiae is a commonly usedlower eukaryotic host microorganism.

Suitable host cells for the expression of glycosylated-IL-22 are derivedfrom multicellular organisms. Examples of invertebrate cells includeinsect cells such as Drosophila S2 and Spodoptera Sf9, as well as plantcells. Examples of useful mammalian host cell lines include Chinesehamster ovary (CHO) and COS cells. More specific examples include monkeykidney CV1 cells transformed by SV40 (COS-7, ATCC CRL 1651); humanembryonic kidney cells (293 or 293 cells subcloned for growth insuspension culture, Graham et al., J. Gen Virol., 36:59 (1977)); Chinesehamster ovary cells/-DHFR (CHO, Urlaub and Chasin, Proc. Natl. Acad.Sci. USA, 77:4216 (1980)); mouse sertoli cells (TM4, Mather, Biol.Reprod., 23:243-251 (1980)); human lung cells (W138, ATCC CCL 75); humanliver cells (Hep G2, HB 8065); and mouse mammary tumor cells (MMT060562, ATCC CCL51). The selection of the appropriate host cell isdeemed to be within the skill in the art.

The nucleic acid (e.g., cDNA or genomic DNA) encoding IL-22 can beinserted into a replicable vector for cloning (amplification of the DNA)or for expression. Various vectors are publicly available. The vectorcan, for example, be in the form of a plasmid, cosmid, viral particle,or phage. The appropriate nucleic acid sequence can be inserted into thevector by a variety of procedures. In general, DNA is inserted into anappropriate restriction endonuclease site(s) using techniques known inthe art. Vector components generally include, but are not limited to,one or more of a signal sequence, an origin of replication, one or moremarker genes, an enhancer element, a promoter, and a transcriptiontermination sequence. Construction of suitable vectors containing one ormore of these components employs standard ligation techniques which areknown to the skilled artisan.

The IL-22 polypeptides can be produced recombinantly not only directly,but also as a fusion polypeptide with a heterologous polypeptide, whichcan be a signal sequence or other polypeptide having a specific cleavagesite at the N-terminus of the mature protein or polypeptide, as well asan IL-22 Fc fusion protein. In general, the signal sequence can be acomponent of the vector, or it can be a part of the IL-22 DNA that isinserted into the vector. The signal sequence can be a prokaryoticsignal sequence selected, for example, from the group of the alkalinephosphatase, penicillinase, 1 pp, or heat-stable enterotoxin II leaders.For yeast secretion the signal sequence can be, e.g., the yeastinvertase leader, alpha factor leader (including Saccharomyces andKluyveromyces”—factor leaders, the latter described in U.S. Pat. No.5,010,182), or acid phosphatase leader, the C. albicans glucoamylaseleader (EP 362,179 published 4 Apr. 1990), or the signal described in WO90/13646 published 15 Nov. 1990. In mammalian cell expression, mammaliansignal sequences can be used to direct secretion of the protein, such assignal sequences from secreted polypeptides of the same or relatedspecies, as well as viral secretory leaders.

Both expression and cloning vectors contain a nucleic acid sequence thatenables the vector to replicate in one or more selected host cells. Suchsequences are well known for a variety of bacteria, yeast, and viruses.The origin of replication from the plasmid pBR322 is suitable for mostGram-negative bacteria, the 2: plasmid origin is suitable for yeast, andvarious viral origins (SV40, polyoma, adenovirus, VSV or BPV) are usefulfor cloning vectors in mammalian cells.

Expression and cloning vectors will typically contain a selection gene,also termed a selectable marker. Typical selection genes encode proteinsthat (a) confer resistance to antibiotics or other toxins, e.g.,ampicillin, neomycin, methotrexate, or tetracycline, (b) complementauxotrophic deficiencies, or (c) supply critical nutrients not availablefrom complex media, e.g., the gene encoding D-alanine racemase forBacilli.

An example of suitable selectable markers for mammalian cells is onethat enables the identification of cells competent to take up the IL-22nucleic acid, such as DHFR or thymidine kinase. An appropriate host cellwhen wild-type DHFR is employed is the CHO cell line deficient in DHFRactivity, prepared and propagated as described by Urlaub et al., Proc.Natl. Acad. Sci. USA, 77:4216 (1980). A suitable selection gene for usein yeast is the trp1 gene present in the yeast plasmid YRp7 [see, e.g.,Stinchcomb et al., Nature, 282:39(1979); Kingsman et al., Gene, 7:141(1979); Tschemper et al., Gene, 10:157 (1980)]. The trp1 gene provides aselection marker for a mutant strain of yeast lacking the ability togrow in tryptophan, for example, ATCC No. 44076 or PEP4-1 [Jones,Genetics, 85:12 (1977)].

Expression and cloning vectors usually contain a promoter operablylinked to the IL-22 nucleic acid sequence to direct mRNA synthesis.Promoters recognized by a variety of potential host cells are wellknown. Promoters suitable for use with prokaryotic hosts include thequadrature-lactamase and lactose promoter systems [see, e.g., Chang etal., Nature, 275:615 (1978); Goeddel et al., Nature, 281:544 (1979)],alkaline phosphatase, a tryptophan (trp) promoter system [see, e.g.,Goeddel, Nucleic Acids Res., 8:4057 (1980); EP 36,776], and hybridpromoters such as the tac promoter [see, e.g., deBoer et al., Proc.Natl. Acad. Sci. USA, 80:21-25 (1983)]. Promoters for use in bacterialsystems also will contain a Shine-Dalgarno (S.D.) sequence operablylinked to the DNA encoding IL-22.

Examples of suitable promoter sequences for use with yeast hosts includethe promoters for 3-phosphoglycerate kinase [see, e.g., Hitzeman et al.,J. Biol. Chem, 255:2073 (1980)] or other glycolytic enzymes [see, e.g.,Hess et al., J. Adv. Enzyme Reg., 7:149 (1968); Holland, Biochemistry,17:4900 (1978)], such as enolase, glyceraldehyde-3-phosphatedehydrogenase, hexokinase, pyruvate decarboxylase, phosphofructokinase,glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvatekinase, triosephosphate isomerase, phosphoglucose isomerase, andglucokinase.

Other yeast promoters, which are inducible promoters having theadditional advantage of transcription controlled by growth conditions,are the promoter regions for alcohol dehydrogenase 2, isocytochrome C,acid phosphatase, degradative enzymes associated with nitrogenmetabolism, metallothionein, glyceraldehyde-3-phosphate dehydrogenase,and enzymes responsible for maltose and galactose utilization. Suitablevectors and promoters for use in yeast expression are further describedin EP 73,657.

IL-22 transcription from vectors in mammalian host cells is controlled,for example, by promoters obtained from the genomes of viruses such aspolyoma virus, fowlpox virus (UK 2,211,504 published 5 Jul. 1989),adenovirus (such as Adenovirus 2), bovine papilloma virus, avian sarcomavirus, cytomegalovirus, a retrovirus, hepatitis-B virus and Simian Virus40 (SV40), from heterologous mammalian promoters, e.g., the actinpromoter or an immunoglobulin promoter, and from heat-shock promoters,provided such promoters are compatible with the host cell systems.

Transcription of a DNA encoding the IL-22 polypeptides by highereukaryotes can be increased by inserting an enhancer sequence into thevector. Enhancers are cis-acting elements of DNA, usually about from 10to 300 bp, that act on a promoter to increase its transcription. Manyenhancer sequences are now known from mammalian genes (globin, elastase,albumin, α-fetoprotein, and insulin). Typically, however, one will usean enhancer from a eukaryotic cell virus. Examples include the SV40enhancer on the late side of the replication origin (bp 100-270), thecytomegalovirus early promoter enhancer, the polyoma enhancer on thelate side of the replication origin, and adenovirus enhancers. Theenhancer can be spliced into the vector at a position 5′ or 3′ to theIL-22 coding sequence, but is preferably located at a site 5′ from thepromoter.

Expression vectors used in eukaryotic host cells (yeast, fungi, insect,plant, animal, human, or nucleated cells from other multicellularorganisms) will also contain sequences necessary for the termination oftranscription and for stabilizing the mRNA. Such sequences are commonlyavailable from the 5′ and, occasionally 3′, untranslated regions ofeukaryotic or viral DNAs or cDNAs. These regions contain nucleotidesegments transcribed as polyadenylated fragments in the untranslatedportion of the mRNA encoding IL-22.

Still other methods, vectors, and host cells suitable for adaptation tothe synthesis of IL-22 in recombinant vertebrate cell culture aredescribed in Gething et al., Nature, 293:620-625 (1981); Mantei et al.,Nature, 281:4046 (1979); EP 117,060; and EP 117,058.

Gene amplification and/or expression can be measured in a sampledirectly, for example, by conventional Southern blotting, Northernblotting to quantitate the transcription of mRNA [see, e.g., Thomas,Proc. Natl. Acad. Sci. USA, 77:5201-5205 (1980)], dot blotting (DNAanalysis), or in situ hybridization, using an appropriately labeledprobe, based on the sequences provided herein. Alternatively, antibodiescan be employed that can recognize specific duplexes, including DNAduplexes, RNA duplexes, and DNA-RNA hybrid duplexes or DNA-proteinduplexes. The antibodies in turn can be labeled and the assay can becarried out where the duplex is bound to a surface, so that upon theformation of duplex on the surface, the presence of antibody bound tothe duplex can be detected.

Gene expression, alternatively, can be measured by immunologicalmethods, such as immunohistochemical staining of cells or tissuesections and assay of cell culture or body fluids, to quantitatedirectly the expression of gene product. Antibodies useful forimmunohistochemical staining and/or assay of sample fluids can be eithermonoclonal or polyclonal, and can be prepared in any mammal.Conveniently, the antibodies can be prepared against a native sequenceIL-22 polypeptide or against a synthetic peptide based on the DNAsequences provided herein or against exogenous sequence fused to IL-22DNA and encoding a specific antibody epitope.

Forms of IL-22 can be recovered from culture medium or from host celllysates. If membrane-bound, it can be released from the membrane using asuitable detergent solution (e.g. Triton-X 100) or by enzymaticcleavage. Cells employed in expression of IL-22 can be disrupted byvarious physical or chemical means, such as freeze-thaw cycling,sonication, mechanical disruption, or cell lysing agents.

It may be desired to purify IL-22 from recombinant cell proteins orpolypeptides. The following procedures are exemplary of suitablepurification procedures: by fractionation on an ion-exchange column;ethanol precipitation; reverse phase HPLC; chromatography on silica oron a cation-exchange resin such as DEAE; chromatofocusing; SDS-PAGE;ammonium sulfate precipitation; gel filtration using, for example,Sephadex G-75; protein A Sepharose columns to remove contaminants suchas IgG; and metal chelating columns to bind epitope-tagged forms of theIL-22 polypeptide. Various methods of protein purification may beemployed and such methods are known in the art and described for examplein Deutscher, Methods in Enzymology, 182 (1990); Scopes, ProteinPurification: Principles and Practice, Springer-Verlag, New York (1982).The purification step(s) selected will depend, for example, on thenature of the production process used and the particular IL-22 produced.The above-described general methods can be applied to the preparation ofIL-2 Fc fusion protein as well.

Similarly, IL-22 Fc fusion proteins may be produced using recombinantmethods and compositions, as described in, e.g., Molecular Cloning: ALaboratory Manual (Sambrook, et al., 1989, Cold Spring Harbor LaboratoryPress) and PCR Protocols: A Guide to Methods and Applications (Innis, etal. 1990. Academic Press, San Diego, Calif.). In one embodiment,isolated nucleic acid encoding IL-22 Fc fusion proteins described hereinis provided. In a further embodiment, one or more vectors (e.g.,expression vectors) comprising such nucleic acid are provided. In afurther embodiment, a host cell comprising such nucleic acid isprovided. In one such embodiment, a host cell comprises (e.g., has beentransformed with) a vector comprising a nucleic acid that encodes anamino acid sequence comprising the IL-22 Fc fusion protein. In certainembodiment, the vector is an expression vector. In one embodiment, thehost cell is eukaryotic, e.g. a Chinese Hamster Ovary (CHO) cell orlymphoid cell (e.g., Y0, NS0, Sp20 cell). In one embodiment, a method ofmaking an IL-22 Fc fusion protein is provided, wherein the methodcomprises culturing a host cell comprising a nucleic acid encoding theIL-22 Fc fusion protein, as provided above, under conditions suitablefor expression of the Fc fusion protein, and optionally recovering theFc fusion protein from the host cell (or host cell culture medium).

For recombinant production of an IL-22 Fc fusion protein, nucleic acidencoding an Fc fusion protein, e.g., as described herein, is isolatedand inserted into one or more vectors for further cloning and/orexpression in a host cell. Such nucleic acid may be readily isolated andsequenced using conventional procedures (e.g., by using oligonucleotideprobes that are capable of binding specifically to genes encoding thefusion protein). In certain embodiments, when preparing the IL-22 Fcfusion proteins, nucleic acid encoding the IL-22 polypeptide or afragment thereof can be ligated to nucleic acid encoding animmunoglobulin constant domain sequence at specified location on theconstant domain to result in an Fc fusion at the C-terminus of IL-22;however N-terminal fusions are also possible.

As an example of constructing an IL-22 Fc fusion protein, the DNAencoding IL-22 is cleaved by a restriction enzyme at or proximal to the3′ end of the DNA encoding IL-22 and at a point at or near the DNAencoding the N-terminal end of the mature polypeptide (where use of adifferent leader is contemplated) or at or proximal to the N-terminalcoding region for IL-22 full-length protein (where a native signal isemployed). This DNA fragment then is readily inserted into DNA encodingan immunoglobulin light or heavy chain constant region and, ifnecessary, tailored by deletional mutagenesis. Preferably, this is ahuman immunoglobulin when the fusion protein is intended for in vivotherapy for humans.

In some embodiments, the IL-22-immunoglobulin chimeras are assembled asmonomers, hetero- or homo-multimer, or as dimers or tetramers.Generally, these assembled immunoglobulins will have known unitstructures as represented by the following diagrams. A basic four chainstructural unit is the form in which IgG, IgD, and IgE exist. A fourchain unit is repeated in the higher molecular weight immunoglobulins;IgM generally exists as a pentamer of, basic four-chain units heldtogether by disulfide bonds. IgA globulin, and occasionally IgGglobulin, may also exist in a multimeric form in serum. In the case ofmultimers, each four chain unit may be the same or different. See alsoCapon et al. U.S. Pat. No. 5,116,964, incorporated herein by referencein its entirety.

In the diagrams herein, “A” means at least a portion of a bindingpartner (such as IL-22) containing a binding site which is capable ofbinding its ligand or receptor (such as IL-22 R); X is an additionalagent, which may be another functional binding partner (same as A ordifferent), a multiple subunit (chain) polypeptide as defined above(e.g., an integrin), a portion of an immunoglobulin superfamily membersuch as a variable region or a variable region-like domain, including anative or chimeric immunoglobulin variable region, a toxin such aspseudomonas exotoxin or ricin, or a polypeptide therapeutic agent nototherwise normally associated with a constant domain; and V_(L), V_(H),C_(L) and C_(H) represent light or heavy chain variable or constantdomains of an immunoglobulin. These diagrams are understood to be merelyexemplary of general assembled immunoglobulin structures, and do notencompass all possibilities. It will be understood, for example, thatthere might desirably be several different “A”s or “X”s in any of theseconstructs.

It will be understood that these diagrams are merely illustrative, andthat the chains of the multimers are believed to be disulfide bonded inthe same fashion as native immunoglobulins. According to this invention,hybrid immunoglobulins are readily secreted from mammalian cellstransformed with the appropriate nucleic acid. The secreted formsinclude those wherein the binding partner epitope is present in heavychain dimers, light chain monomers or dimers, and heavy and light chainheterotetramers wherein the binding partner epitope is present fused toone or more light or heavy chains, including heterotetramers wherein upto and including all four variable region analogues are substituted.Where a light-heavy chain non-binding partner variable-like domain ispresent, a heterofunctional antibody thus is provided.

Chains or basic units of varying structure may be utilized to assemblethe monomers and hetero- and homo-multimers and immunoglobulins of thisinvention. Specific examples of these basic units are diagrammed belowand their equivalents (for purposes of the attenuated formulae infra)are indicated.

Various exemplary assembled novel immunoglobulins produced in accordancewith this invention are schematically diagrammed below. In addition tothe symbols defined above, n is an integer, and Y designates a covalentcross-linking moiety.

-   -   (a) AC_(L);    -   (b) AC_(L)-AC_(L);    -   (c) AC_(H)-[AC_(H), AC_(L)-AC_(H), AC_(L)-V_(H)C_(H),        V_(L)C_(L)-AC_(H), V_(L)C_(L)-V_(H)C_(H), XC_(H), XC_(L),        XC_(L)-XC_(H), XC_(L)-V_(H)C_(H), XC_(H)-V_(L)C_(L),        XC_(L)-AC_(H) or AC_(L)-XC_(H)];    -   (d) AC_(L)-AC_(H)-[AC_(H), AC_(L)-AC_(H), AC_(L)-V_(H)C_(H),        V_(L)C_(L)-AC_(H), V_(L)C_(L)-V_(H)C_(H), XC_(H), XC_(L),        XC_(L)-XC_(H), XC_(L)-V_(H)C_(H), XC_(H)-V_(L)C_(L),        XC_(L)-AC_(H), or AC_(L)-XC_(H)];    -   (e) AC_(L)-V_(H)C_(H)-[AC_(H), AC_(L)-AC_(H), AC_(L)-V_(H)C_(H),        V_(L)C_(L)-AC_(H), V_(L)C_(L)-V_(H)C_(H), XC_(H), XC_(L),        XC_(L)-XC_(H), XC_(L)-V_(H)C_(H), XC_(H)-V_(L)C_(L),        XC_(L)-AC_(H), or AC_(L)-XC_(H)];    -   (f) V_(L)C_(L)-AC_(H)-[AC_(H), AC_(L)-AC_(H), AC_(L)-V_(H)C_(H),        V_(L)C_(L)-AC_(H), V_(L)C_(L)-V_(H)C_(H), XC_(H), XC_(L),        XC_(L)-XC_(H), XC_(L)-V_(H)C_(H), XC_(H)-V_(L)C_(L),        XC_(L)-AC_(H), or AC_(L)-XC_(H)];    -   (g) [A-Y]_(n)-[V_(L)C_(L)-V_(H)C_(H)]₂;    -   (h) XC_(H) or XC_(L)-[AC_(H), AC_(L)-AC_(H), AC_(L)-V_(H)C_(H),        V_(L)C_(L)-AC_(H), XC_(L)-AC_(H), or AC_(L)-XC_(H)];    -   (i) XC_(L)-XC_(H)-[AC_(H), AC_(L)-AC_(H), AC_(L)-V_(H)C_(H),        V_(L)C_ACH , XC_(L)-AC_(H), or AC_(L)-XC_(H)];    -   (j) XC_(L)-V_(H)C_(H)-[AC_(H), AC_(L)-AC_(H), AC_(L)-V_(H)C_(H),        V_(L)C_(L)-AC_(H), XC_(L)-AC_(H), or AC_(L)-XC_(H)]2    -   (k) XC_(H)-V_(L)C_(L)-[AC_(H), AC_(L)-AC_(H), AC_(L)-V_(H)C_(H),        V_(L)C_(L)-AC_(H), XC_(L)-AC_(H), or AC_(L)-XC_(H)];    -   (l) XC_(L)-AC_(H)-[AC_(H), AC_(L)-AC_(H), AC_(L)-V_(H)C_(H),        V_(L)C_(L)-AC_(H), V_(L)C_(L)-V_(H)C_(H), XC_(H), XC_(L),        XC_(L)-XC_(H), XC_(L)-V_(H)C_(H), XC_(H)-V_(L)C_(L),        XC_(L)-AC_(H), or AC_(L)-XC_(H)];    -   (m) AC_(L)-XC_(H)-[AC_(H), AC_(L)-AC_(H), AC_(L)-V_(H)C_(H),        V_(L)C_(L)-AC_(H), V_(L)C_(L)-V_(H)C_(H), XC_(H), XC_(L),        XC_(L)-XC_(H), XC_(L)-V_(H)C_(H), XC_(H)-V_(L)C_(L),        XC_(L)-AC_(H), or AC_(L)-XC_(H)];

A, X, V or C may be modified with a covalent cross-linking moiety (Y) soto be (A-Y)_(n), (X-Y)_(n) etc.

The binding partner A (such as IL-22) may also be a multi-chainmolecule, e.g. having chains arbitrarily denoted as A_(a) and A_(β).These chains as a unit are located at the sites noted for the singlechain “A” above. One of the multiple chains is fused to oneimmunoglobulin chain (with the remaining chains covalently ornoncovalently associated with the fused chain in the normal fashion) or,when the ligand binding partner contains two chains, one chain isseparately fused to an immunoglobulin light chain and the other chain toan immunoglobulin heavy chain.

Basic units having the structures as diagrammed below are examples ofthose used to create monomers, and hetero- and homo-multimers,particularly dimers and trimers with multi-chain ligand bindingpartners:

Various exemplary novel assembled antibodies having a two-chain ligandbinding partner (“A_(α) and A_(β)”) utilized in unit structures as aboveare schematically diagrammed below.

-   -   (n) A_(α)A_(β)C_(L)-[AC_(H), AC_(L)-AC_(H), AC_(L)-V_(H)C_(H),        V_(L)C_(L)-AC_(H), V_(L)C_(L)-V_(H)C_(H), XC_(H), XC_(L),        XC_(L)-XC_(H), XC_(L)-V_(H)C_(H), XC_(H)-V_(L)C_(L),        XC_(L)-AC_(H), AC_(L)-XC_(H), A_(α)AβC_(H), A_(α)A_(β)C_(L),        AαC_(L)-AαC_(H), A_(β)C_(L)-AαC_(H), A_(α)AβC_(L)-V_(H)C_(H),        A_(α)AβC_(H)-V_(L)C_(L), A_(α)A_(β)C_(L)-XC_(H), or        A_(α)AβC_(H)-XC_(L)];    -   (o) A_(α)AβC_(H)-[AC_(H), AC_(L)-AC_(H), AC_(L)-V_(H)C_(H),        V_(L)C_(L)-AC_(H), V_(L)C_(L)-V_(H)C_(H), XC_(H), XC_(L),        XC_(L)-XC_(H), XC_(L)-V_(H)C_(H), XC_(H)-V_(L)C_(L),        XC_(L)-AC_(H), AC_(L)-XC_(H), A_(α)AβC_(H), A_(α)A_(β)C_(L),        AαC_(L)-AβC_(H), A_(β)C_(L)-AαC_(H), A_(α)A_(β)C_(L)-V_(H)C_(H),        A_(α)AβC_(H)-V_(L)C_(L), A_(α)A_(β)C_(L)-XC_(L), or        A_(α)AβC_(H)-XC_(L)];    -   (p) A_(α)C_(L)-AβC_(H)-[AC_(H), AC_(L)-AC_(H),        AC_(L)-V_(H)C_(H), V_(L)C_(L)-AC_(H), V_(L)C_(L)-V_(H)C_(H),        XC_(H), XC_(L), XC_(L)-XC_(H), XC_(L)-V_(H)C_(H),        XC_(H)-V_(L)C_(L), XC_(L)-AC_(H), AC_(L)-XC_(H), A_(α)AβC_(H),        A_(α)A_(β)C_(L), AαC_(L)-AβC_(H), A_(β)C_(L)-AαC_(H),        A_(α)AβC_(L)-V_(H)C_(H), A_(α)AβC_(H)-V_(L)C_(L),        A_(α)A_(β)C_(L)-XC_(H), or A_(α)AβC_(H)-XC_(L)];    -   (q) A_(β)C_(L)-AαC_(H)-[AC_(H), AC_(L)-AC_(H),        AC_(L)-V_(H)C_(H), V_(L)C_(L)-AC_(H), V_(L)C_(L)-V_(H)C_(H),        XC_(H), XC_(L), XC_(L)-XC_(H), XC_(L)-V_(H)C_(H),        XC_(H)-V_(L)C_(L), XC_(L)-AC_(H), AC_(L)-XC_(H), A_(α)AβC_(H),        A_(α)A_(β)C_(L), AαC_(L)-AβC_(H), A_(β)C_(L)-AαC_(H),        A_(α)AβC_(L)-V_(H)C_(H), A_(α)AβC_(H)-V_(L)C_(L),        A_(α)A_(β)C_(L)-XC_(H), or A_(α)AβC_(H)-XC_(L)];    -   (r) A_(α)AβC_(L)-V_(H)C_(H)-[AC_(H), AC_(L)-AC_(H),        AC_(L)-V_(H)C_(H), V_(L)C_(L)-AC_(H), V_(L)C_(L)-V_(H)C_(H),        XC_(H), XC_(L), XC_(L)-XC_(H), XC_(L)-V_(H)C_(H),        XC_(H)-V_(L)C_(L), XC_(L)-AC_(H), AC_(L)-XC_(H), A_(α)AβC_(H),        A_(α)A_(β)C_(L), AαC_(L)-AβC_(H), A_(β)C_(L)-AαC_(H),        A_(α)AβC_(L)-V_(H)C_(H), A_(α)AβC_(H)-V_(L)C_(L),        A_(α)A_(β)C_(L)-XC_(H), or A_(α)AβC_(H)-XC_(L)];    -   (s) A_(α)AβC_(H)-V_(L)C_(L)-[AC_(H), AC_(L)-AC_(H),        AC_(L)-V_(H)C_(H), V_(L)C_(L)-AC_(H), V_(L)C_(L)-V_(H)C_(H),        XC_(H), XC_(L), XC_(L)-XC_(H), XC_(L)-V_(H)C_(H),        XC_(H)-V_(L)C_(L), XC_(L)-AC_(H), AC_(L)-XC_(H), A_(α)AβC_(H),        A_(α)A_(β)C_(L), AαC_(L)-AβC_(H), A_(β)C_(L)-AαC_(H),        A_(α)AβC_(L)-V_(H)C_(H), A_(α)AβC_(H)-V_(L)C_(L),        A_(α)A_(β)C_(L)-XC_(H), or A_(α)AβC_(H)-XC_(L)];    -   (t) A_(α)AβC_(L)-XC_(H)-[AC_(H), AC_(L)-AC_(H),        AC_(L)-V_(H)C_(H), V_(L)C_(L)-AC_(H), V_(L)C_(L)-V_(H)C_(H),        XC_(H), XC_(L), XC_(L)-XC_(H), XC_(L)-V_(H)C_(H),        XC_(H)-V_(L)C_(L), XC_(L)-AC_(H), AC_(L)-XC_(H), A_(α)AβC_(H),        A_(α)A_(β)C_(L), AαC_(L)-AβC_(H), A_(β)C_(L)-AαC_(H),        A_(α)AβC_(L)-V_(H)C_(H), A_(α)AβC_(H)-V_(L)C_(L),        A_(α)A_(β)C_(L)-XC_(H), or A_(α)AβC_(H)-XC_(L)];    -   (u) A_(α)AβC_(H)-[AC_(H), AC_(L)-AC_(H), AC_(L)-V_(H)C_(H),        V_(L)C_(L)-AC_(H), V_(L)C_(L)-V_(H)C_(H), XC_(H), XC_(L),        XC_(L)-XC_(H), XC_(L)-V_(H)C_(H), XC_(H)-V_(L)C_(L),        XC_(L)-AC_(H), AC_(L)-XC_(H), A_(α)AβC_(H), A_(α)A_(β)C_(L),        AαC_(L)-AβC_(H), A_(β)C_(L)-AαC_(H), A_(α)AβC_(L)-V_(H)C_(H),        A_(α)AβC_(H)-V_(L)C_(L), A_(α)AβC-XC_(H), or        A_(α)AβC_(H)-XC_(L)].

The structures shown in the above tables show only key features, e.g.they do not show joining (J) or other domains of the immunoglobulins,nor are disulfide bonds shown. These are omitted in the interests ofbrevity. However, where such domains are required for binding activitythey shall be constructed as being present in the ordinary locationswhich they occupy in the binding partner or immunoglobulin molecules asthe case may be.

DNA encoding immunoglobulin light or heavy chain constant regions isknown or readily available from cDNA libraries or is synthesized. Seefor example, Adams et al., Biochemistry 19:2711-2719 (1980); Gough etal., Biochemistry 19:2702-2710 (1980); Dolby et al; P.N.A.S. USA,77:6027-6031 (1980); Rice et al P.N.A.S USA 79:7862-7865 (1982); Falkneret al; Nature 298:286-288 (1982); and Morrison et al; Ann. Rev. Immunol.2:239-256 (1984). DNA sequence encoding human IL-22 with the endogenousleader sequence is provided herein (SEQ ID NO:70). DNA sequencesencoding other desired binding partners which are known or readilyavailable from cDNA libraries are suitable in the practice of thisinvention.

DNA encoding an IL-22 Fc fusion protein of this invention is transfectedinto a host cell for expression. If multimers are desired then the hostcell is transformed with DNA encoding each chain that will make up themultimer, with the host cell optimally being selected to be capable ofassembling the chains of the multimers in the desired fashion. If thehost cell is producing an immunoglobulin prior to transfection then oneneeds only transfect with the binding partner fused to light or to heavychain to produce a heteroantibody. The aforementioned immunoglobulinshaving one or more arms bearing the binding partner domain and one ormore arms bearing companion variable regions result in dual specificityfor the binding partner ligand and for an antigen or therapeutic moiety.Multiply cotransformed cells are used with the above-describedrecombinant methods to produce polypeptides having multiplespecificities such as the heterotetrameric immunoglobulins discussedabove.

Although the presence of an immunoglobulin light chain is not requiredin the immunoadhesins of the present invention, an immunoglobulin lightchain might be present either covalently associated to anIL-22-immunoglobulin heavy chain fusion polypeptide. In this case, DNAencoding an immunoglobulin light chain is typically co-expressed withthe DNA encoding the IL-22-immunoglobulin heavy chain fusion protein.Upon secretion, the hybrid heavy chain and the light chain will becovalently associated to provide an immunoglobulin-like structurecomprising two disulfide-linked immunoglobulin heavy chain-light chainpairs. Methods suitable for the preparation of such structures are, forexample, disclosed in U.S. Pat. No. 4,816,567 issued Mar. 28, 1989.Suitable host cells for cloning or expression of target protein-encodingvectors include prokaryotic or eukaryotic cells described herein. Forexample, IL-22 fusion protein may be produced in bacteria, in particularwhen glycosylation and Fc effector function are not needed or aredetrimental. For expression of polypeptides in bacteria, see, e.g., U.S.Pat. Nos. 5,648,237, 5,789,199, and 5,840,523. (See also Charlton,Methods in Molecular Biology, Vol. 248 (B. K. C. Lo, ed., Humana Press,Totowa, N.J., 2003), pp. 245-254, describing expression of antibodyfragments in E. coli.) After expression, the Fc fusion protein may beisolated from the bacterial cell paste in a soluble fraction and can befurther purified. As exemplified in the example section, furtherpurification methods include without limitation purification using aProtein A column.

In addition to prokaryotes, eukaryotic microbes such as filamentousfungi or yeast are suitable cloning or expression hosts, including fungiand yeast strains whose glycosylation pathways have been “humanized,”resulting in the production of an antibody with a partially or fullyhuman glycosylation pattern. See Gerngross, Nat. Biotech. 22:1409-1414(2004), and Li et al., Nat. Biotech. 24:210-215 (2006).

Suitable host cells for the expression of glycosylated proteins are alsoderived from multicellular organisms (invertebrates and vertebrates).Examples of invertebrate cells include plant and insect cells. Numerousbaculoviral strains have been identified which may be used inconjunction with insect cells, particularly for transfection ofSpodoptera frugiperda cells.

Plant cell cultures can also be utilized as hosts. See, e.g., U.S. Pat.Nos. 5,959,177, 6,040,498, 6,420,548, 7,125,978, and 6,417,429(describing PLANTIBODIES™ technology for producing antibodies intransgenic plants).

Vertebrate cells may also be used as hosts. For example, mammalian celllines that are adapted to grow in suspension may be useful. Otherexamples of useful mammalian host cell lines are monkey kidney CV1 linetransformed by SV40 (COS-7); human embryonic kidney line (293 or 293cells as described, e.g., in Graham et al., J. Gen Virol. 36:59 (1977));baby hamster kidney cells (BHK); mouse sertoli cells (TM4 cells asdescribed, e.g., in Mather, Biol. Reprod. 23:243-251 (1980)); monkeykidney cells (CV1); African green monkey kidney cells (VERO-76); humancervical carcinoma cells (HELA); canine kidney cells (MDCK; buffalo ratliver cells (BRL 3A); human lung cells (W138); human liver cells (HepG2); mouse mammary tumor (MMT 060562); TRI cells, as described, e.g., inMather et al., Annals N.Y. Acad. Sci. 383:44-68 (1982); MRC 5 cells; andFS4 cells. Other useful mammalian host cell lines include Chinesehamster ovary (CHO) cells, including DHFR⁻ CHO cells (Urlaub et al.,Proc. Natl. Acad. Sci. USA 77:4216 (1980)); and myeloma cell lines suchas Y0, NS0 and Sp2/0. For a review of certain mammalian host cell linessuitable for antibody production, see, e.g., Yazaki and Wu, Methods inMolecular Biology, Vol. 248 (B. K. C. Lo, ed., Humana Press, Totowa,N.J.), pp. 255-268 (2003).

D. IL-22 Agonists

In one aspect, the present invention provides IL-22 agonists for methodembodiments. The IL-22 agonists have IL-22 biological activity asdefined herein. In one embodiment, the IL-22 agonist is an antibody. Incertain embodiments, an anti-IL-22 antibody is an agonistic antibodythat promotes the interaction of IL-22 with IL-22R. In a particularembodiment, an IL-22 agonist is an antibody that binds IL-22BP andblocks or inhibits binding of IL-22BP to IL-22, and thereby induces orincreases an IL-22 activity (e.g., binding to IL-22R). In anotherembodiment, an IL-22 agonist is an oligopeptide that binds to IL-22.Oligopeptides can be chemically synthesized using known oligopeptidesynthesis methodology or can be prepared and purified using recombinanttechnology. Such oligopeptides are usually at least about 5 amino acidsin length, alternatively at least about 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32,33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50,51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68,69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86,87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 amino acidsin length. Such oligopeptides can be identified without undueexperimentation using well known techniques. In this regard, it is notedthat techniques for screening oligopeptide libraries for oligopeptidesthat are capable of specifically binding to a polypeptide target arewell known in the art (see, e.g., U.S. Pat. Nos. 5,556,762, 5,750,373,4,708,871, 4,833,092, 5,223,409, 5,403,484, 5,571,689, 5,663,143; PCTPublication Nos. WO 84/03506 and WO84/03564; Geysen et al., Proc. Natl.Acad. Sci. U.S.A., 81:3998-4002 (1984); Geysen et al., Proc. Natl. Acad.Sci. USA, 82:178-182 (1985); Geysen et al., in Synthetic Peptides asAntigens, 130-149 (1986); Geysen et al., J. Immunol. Meth., 102:259-274(1987); Schoofs et al., J. Immunol., 140:611-616 (1988), Cwirla, S. E.et al. (1990) Proc. Natl. Acad. Sci. USA, 87:6378; Lowman, H. B. et al.(1991) Biochemistry, 30:10832; Clackson, T. et al. (1991) Nature, 352:624; Marks, J. D. et al. (1991), J. Mol. Biol., 222:581; Kang, A. S. etal. (1991) Proc. Natl. Acad. Sci. USA, 88:8363, and Smith, G. P. (1991)Current Opin. Biotechnol., 2:668).

In yet another embodiment, an IL-22 agonist of the present invention isan organic molecule that binds to IL-22, other than an oligopeptide orantibody as described herein. An organic molecule can be, for example, asmall molecule. An organic molecule that binds to IL-22 can beidentified and chemically synthesized using known methodology (see,e.g., PCT Publication Nos. WO00/00823 and WO00/39585). Such organicmolecules are usually less than about 2000 daltons in size,alternatively less than about 1500, 750, 500, 250 or 200 daltons insize, wherein such organic molecules that are capable of binding toIL-22 of the present invention can be identified without undueexperimentation using well known techniques. In this regard, it is notedthat techniques for screening organic molecule libraries for moleculesthat are capable of binding to a polypeptide target are well known inthe art (see, e.g., PCT Publication Nos. WO00/00823 and WO00/39585). Ina particular embodiment, an IL-22 agonist is an organic molecule thatbinds IL-22BP and blocks or inhibits binding of IL-22BP to IL-22, andthereby induces or increases an IL-22 activity (e.g., binding toIL-22R). In yet another embodiment, agonists of IL-22 are provided.Exemplary agonists include, but are not limited to, native IL-22 orIL-22R; fragments, variants, or modified forms of IL-22 or IL-22R thatretain at least one activity of the native polypeptide; agents that areable to bind to and activate IL-22R; and agents that induceover-expression of IL-22 or IL-22R or nucleic acids encoding IL-22 orIL-22R.

E. Assays

IL-22 Fc fusion protein provided herein may be identified, screened for,or characterized for their physical/chemical properties and/orbiological activities by various assays known in the art.

1. Binding Assays and Other Assays

In one aspect, an IL-22 Fc fusion protein of the invention is tested forits receptor binding activity, e.g., by known methods such as ELISA,western blotting analysis, cell surface binding by Scatchard, surfaceplasmon resonance. In another aspect, competition assays may be used toidentify an antibody that competes with the IL-22 Fc fusion protein forbinding to the IL-22 receptor. In a further aspect, an IL-22 Fc fusionprotein of the invention can be used for detecting the presence oramount of IL-22 receptor or 11,22-Binding Protein (soluble receptor)present in a biological sample. In a further aspect, an IL-22 Fc fusionprotein of the invention can be used for detecting the presence oramount of IL-22 receptor present in a biological sample. In certainembodiments, the biological sample is first blocked with a non-specificisotype control antibody to saturate any Fc receptors in the sample.

2. Activity Assays

In one aspect, assays are provided for identifying biological activityof IL-22 Fc fusion protein. Biological activity of an IL-22 polypeptideor IL-22 Fc fusion protein may include, e.g., binding to IL-22 receptor,stimulating IL-22 signaling, and inducing STAT3, RegIII and/or PancrePAPexpression. Further, in the case of a cardiovascular disease orcondition, the biological activity may include affecting the formationof atherosclerotic plaques, in particular to inhibit formation ofatherosclerotic plaque formation. Inhibition of plaque formation can beassessed by any suitable imaging method known to those of ordinary skillin the art.

F. Conjugates

The invention also provides conjugates comprising an IL-22 Fc fusionprotein described herein conjugated to one or more agents for detection,formulation, half-life extension, mitigating immunogenicity or tissuepenetration. Exemplary conjugation includes without limitationPEGylation and attaching to radioactive isotopes.

In another embodiment, a conjugate comprises an IL-22 Fc fusion proteinas described herein conjugated to a radioactive atom to form aradioconjugate. A variety of radioactive isotopes are available for theproduction of radioconjugates. Examples include At²¹¹, I¹³¹, I¹²⁵, Y⁹⁰,Re¹⁸⁶, Re¹⁸⁸, Sm¹⁵³, Bi²¹², P³², Pb²¹² and radioactive isotopes of Lu.When the radioconjugate is used for detection, it may comprise aradioactive atom for scintigraphic studies, for example tc99m or I123,or a spin label for nuclear magnetic resonance (NMR) imaging (also knownas magnetic resonance imaging, mri), such as iodine-123 again,iodine-131, indium-111, fluorine-19, carbon-13, nitrogen-15, oxygen-17,gadolinium, manganese or iron.

G. Methods and Compositions for Detection

In certain embodiments, any of the IL-22 Fc fusion provided herein isuseful for detecting the presence of IL-22 receptor in a biologicalsample. In certain embodiments, the method further comprises the step ofblocking any Fc receptors in the sample with a non-specific isotypecontrol antibody. The term “detecting” as used herein encompassesquantitative or qualitative detection. In certain embodiments, abiological sample comprises a cell or tissue, such as epithelialtissues.

In one embodiment, an IL-22 Fc fusion protein for use in a method ofdetection is provided. In a further aspect, a method of detecting thepresence of IL-22 receptor in a biological sample is provided. Incertain embodiments, the method comprises contacting the biologicalsample with an IL-22 Fc fusion protein as described herein underconditions permissive for binding of the IL-22 Fc fusion protein toIL-22 receptor, and detecting whether a complex is formed between theIL-22 Fc fusion protein and IL-22 receptor. In certain embodiments, themethod further comprises the step of blocking any Fc receptors in thesample with a non-specific isotype control antibody. Such method may bean in vitro or in vivo method. In one embodiment, an IL-22 Fc fusionprotein is used to select subjects eligible for therapy with IL-22 Fcfusion protein, e.g. where IL-22 receptor is a biomarker for selectionof patients.

In certain embodiments, labeled IL-22 Fc fusion proteins are provided.Labels include, but are not limited to, labels or moieties that aredetected directly (such as fluorescent, chromophoric, electron-dense,chemiluminescent, and radioactive labels), as well as moieties, such asenzymes or ligands, that are detected indirectly, e.g., through anenzymatic reaction or molecular interaction. Exemplary labels include,but are not limited to, the radioisotopes ³²P, ¹⁴C, ¹²⁵I, ³H, and ¹³¹I,fluorophores such as rare earth chelates or fluorescein and itsderivatives, rhodamine and its derivatives, dansyl, umbelliferone,luceriferases, e.g., firefly luciferase and bacterial luciferase (U.S.Pat. No. 4,737,456), luciferin, 2,3-dihydrophthalazinediones,horseradish peroxidase (HRP), alkaline phosphatase, β-galactosidase,glucoamylase, lysozyme, saccharide oxidases, e.g., glucose oxidase,galactose oxidase, and glucose-6-phosphate dehydrogenase, heterocyclicoxidases such as uricase and xanthine oxidase, coupled with an enzymethat employs hydrogen peroxide to oxidize a dye precursor such as HRP,lactoperoxidase, or microperoxidase, biotin/avidin, spin labels,bacteriophage labels, stable free radicals, and the like.

H. Pharmaceutical Formulations

The IL-22-based compositions (which in certain embodiments, includeIL-22 Fc fusion proteins, and IL-22 polypeptide or agonists) herein willbe formulated, dosed, and administered in a fashion consistent with goodmedical practice. Factors for consideration in this context include theparticular disorder being treated, the particular mammal being treated,the clinical condition of the individual subject, the cause of thedisorder, the site of delivery of the agent, the method ofadministration, the scheduling of administration, and other factorsknown to medical practitioners. In one embodiment, the composition canbe used for increasing the duration of survival of a human subjectsusceptible to or diagnosed with the disease or condition disease.Duration of survival is defined as the time from first administration ofthe drug to death.

Pharmaceutical formulations are prepared using standard methods known inthe art by mixing the active ingredient having the desired degree ofpurity with one or more optional pharmaceutically acceptable carriers(Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)and Remington's Pharmaceutical Sciences 20^(th) edition, ed. A. FGennaro, 2000, Lippincott, Williams & Wilkins, Philadelphia, Pa.), inthe form of lyophilized formulations or aqueous solutions.Pharmaceutically acceptable carriers are generally nontoxic torecipients at the dosages and concentrations employed, and include, butare not limited to: buffers such as phosphate, citrate, and otherorganic acids; antioxidants including ascorbic acid and methionine;preservatives (such as octadecyldimethylbenzyl ammonium chloride;hexamethonium chloride; benzalkonium chloride; benzethonium chloride;phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propylparaben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol);low molecular weight (less than about 10 residues) polypeptides;proteins, such as serum albumin, gelatin, or immunoglobulins;hydrophilic polymers such as polyvinylpyrrolidone; amino acids such asglycine, glutamine, asparagine, histidine, arginine, or lysine;monosaccharides, disaccharides, and other carbohydrates includingglucose, mannose, or dextrins; chelating agents such as EDTA; sugarssuch as sucrose, mannitol, trehalose or sorbitol; salt-formingcounter-ions such as sodium; metal complexes (e.g. Zn-proteincomplexes); and/or non-ionic surfactants such as polyethylene glycol(PEG). Exemplary pharmaceutically acceptable carriers herein furtherinclude insterstitial drug dispersion agents such as solubleneutral-active hyaluronidase glycoproteins (sHASEGP), for example, humansoluble PH-20 hyaluronidase glycoproteins, such as rHuPH20 (HYLENEX®,Baxter International, Inc.). Certain exemplary sHASEGPs and methods ofuse, including rHuPH20, are described in US Patent Publication Nos.2005/0260186 and 2006/0104968. In one aspect, a sHASEGP is combined withone or more additional glycosaminoglycanases such as chondroitinases.

Optionally, but preferably, the formulation contains a pharmaceuticallyacceptable salt, preferably sodium chloride, and preferably at aboutphysiological concentrations.

Optionally, the formulations of the invention can contain apharmaceutically acceptable preservative. In some embodiments thepreservative concentration ranges from 0.1 to 2.0%, typically v/v.Suitable preservatives include those known in the pharmaceutical arts.Benzyl alcohol, phenol, m-cresol, methylparaben, benzalkonium chlorideand propylparaben are preferred preservatives. Optionally, theformulations of the invention can include a pharmaceutically acceptablesurfactant at a concentration of 0.005 to 0.02%.

The formulation herein can also contain more than one active compound asnecessary for the particular indication being treated, preferably thosewith complementary activities that do not adversely affect each other.Such molecules are suitably present in combination in amounts that areeffective for the purpose intended.

Exemplary lyophilized formulations are described in U.S. Pat. No.6,267,958. Aqueous formulations include those described in U.S. Pat. No.6,171,586 and WO2006/044908, the latter formulations including ahistidine-acetate buffer.

The formulation herein may also contain more than one active ingredientsas necessary for the particular indication being treated, preferablythose with complementary activities that do not adversely affect eachother. For example, it may be desirable to further provide a steroid,TNF antagonist or other anti-inflammatory therapeutics. Such activeingredients are suitably present in combination in amounts that areeffective for the purpose intended.

Active ingredients may be entrapped in microcapsules prepared, forexample, by coacervation techniques or by interfacial polymerization,for example, hydroxymethylcellulose or gelatin-microcapsules andpoly-(methylmethacylate) microcapsules, respectively, in colloidal drugdelivery systems (for example, liposomes, albumin microspheres,microemulsions, nano-particles and nanocapsules) or in macroemulsions.Such techniques are disclosed in Remington's Pharmaceutical Sciences16th edition, Osol, A. Ed. (1980).

Sustained-release preparations may be prepared. Suitable examples ofsustained-release preparations include semipermeable matrices of solidhydrophobic polymers containing the IL-22 Fc fusion protein, whichmatrices are in the form of shaped articles, e.g. films, ormicrocapsules. Examples of sustained-release matrices includepolyesters, hydrogels (for example, poly(2-hydroxyethyl-methacrylate),or poly(vinylalcohol)), polylactides (U.S. Pat. No. 3,773,919),copolymers of L-glutamic acid and .gamma. ethyl-L-glutamate,non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolicacid copolymers such as the LUPRON DEPOT™ (injectable microspherescomposed of lactic acid-glycolic acid copolymer and leuprolide acetate),and poly-D-(−)-3-hydroxybutyric acid. While polymers such asethylene-vinyl acetate and lactic acid-glycolic acid enable release ofmolecules for over 100 days, certain hydrogels release proteins forshorter time periods. When encapsulated antibodies remain in the bodyfor a long time, they may denature or aggregate as a result of exposureto moisture at 37° C., resulting in a loss of biological activity andpossible changes in immunogenicity. Rational strategies can be devisedfor stabilization depending on the mechanism involved. For example, ifthe aggregation mechanism is discovered to be intermolecular S—S bondformation through thio-disulfide interchange, stabilization may beachieved by modifying sulfhydryl residues, lyophilizing from acidicsolutions, controlling moisture content, using appropriate additives,and developing specific polymer matrix compositions.

A pharmaceutical composition for topical administration can beformulated, for example, in the form of a topical gel. See e.g., U.S.Pat. Nos. 4,717,717, 5,130,298, 5,427,778, 5,457,093, 5,705,485,6,331,309 and WO2006/138,468. In certain embodiments, the compositioncan be formulated in the presence of cellulose derivatives. In certainother embodiments, the topical formulation can be reconstituted fromlyophilized formulation with sufficient buffer or diluent beforeadministration. In certain embodiments, IL-22 polypeptide or IL-22 Fcfusion protein is formulated for topical administration to a subjecthaving a defect in epithelial wound healing. In certain particularembodiments, the epithelial wound healing occurs in the skin. In certainother particular embodiments, the subject is a human having a defect inwound healing. In certain other embodiments, the topical formulationcomprising an IL-22 Fc fusion protein of the invention can be used toimprove wound healing after internal or external surgical incisions.

In one embodiment of the invention, an IL-22 polypeptide or IL-22 Fcfusion protein for use in accelerating, promoting or improving woundhealing is in a formulation of a topical gel, e.g., in a pre-filledsyringe or container, or alternatively, the compound of the inventioncan be mixed with a gel matrix right before topical administration to apatient. In certain embodiments, an additional therapeutic agent is alsoadministered topically, either concurrently or sequentially. Otherroutes of administration can also be optionally used, e.g., administeredby any suitable means, including but not limited to, parenteral,subcutaneous, intraperitoneal, intrapulmonary, intracerobrospinal,subcutaneous, intra-articular, intrasynovial, intrathecal, oral, andintranasal administration. Parenteral infusions include intramuscular,intravenous, intraarterial, intraperitoneal, or subcutaneousadministration.

Typically for wound healing, an IL-22 polypeptide or IL-22 Fc fusionprotein is formulated for site-specific delivery. When appliedtopically, the IL-22 polypeptide or IL-22 Fc fusion is suitably combinedwith other ingredients, such as carriers and/or adjuvants. There are nolimitations on the nature of such other ingredients, except that theymust be pharmaceutically acceptable and efficacious for their intendedadministration, and cannot degrade the activity of the activeingredients of the composition. Examples of suitable vehicles includeointments, creams, gels, sprays, or suspensions, with or withoutpurified collagen. The compositions also may be impregnated into steriledressings, transdermal patches, plasters, and bandages, optionally inliquid or semi-liquid form. An oxidized regenerated cellulose/collagenmatrices can also be used, e.g., PROMOGRAN Matrix Wound Dressing orPROMOGRAN PRISMA MATRIX.

Sustained-release preparations may be prepared. Suitable examples ofsustained-release preparations include semipermeable matrices of solidhydrophobic polymers containing a polypeptide of the invention, whichmatrices are in the form of shaped articles, e.g. films, ormicrocapsules. Examples of sustained-release matrices includepolyesters, hydrogels (for example, poly(2-hydroxyethyl-methacrylate),or poly(vinylalcohol), polylactides (U.S. Pat. No. 3,773,919),copolymers of L-glutamic acid and gamma ethyl-L-glutamate,non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolicacid copolymers such as the LUPRON DEPOT™ (injectable microspherescomposed of lactic acid-glycolic acid copolymer and leuprolide acetate),poly-lactic-coglycolic acid (PLGA) polymer, andpoly-D-(−)-3-hydroxybutyric acid. While polymers such as ethylene-vinylacetate and lactic acid-glycolic acid enable release of molecules forover 100 days, certain hydrogels release proteins for shorter timeperiods. When encapsulated polypeptides remain in the body for a longtime, they may denature or aggregate as a result of exposure to moistureat 37° C., resulting in a loss of biological activity and possiblechanges in immunogenicity. Rational strategies can be devised forstabilization depending on the mechanism involved. For example, if theaggregation mechanism is discovered to be intermolecular S—S bondformation through thio-disulfide interchange, stabilization may beachieved by modifying sulfhydryl residues, lyophilizing from acidicsolutions, controlling moisture content, using appropriate additives,and developing specific polymer matrix compositions.

For obtaining a gel formulation, the IL-22 polypeptide or IL-22 Fcfusion protein formulated in a liquid composition may be mixed with aneffective amount of a water-soluble polysaccharide or synthetic polymerto form a gel (e.g., a gelling agent) such as polyethylene glycol toform a formulation of the proper viscosity to be applied topically. Thepolysaccharide or gelling agent that may be used includes, for example,cellulose derivatives such as etherified cellulose derivatives,including alkyl celluloses, hydroxyalkyl celluloses, andalkylhydroxyalkyl celluloses, for example, methylcellulose, hydroxyethylcellulose, carboxymethyl cellulose, hydroxypropyl methylcellulose, andhydroxypropyl cellulose; Sodium carboxymethyl cellulose; POE-POP blockpolymers: poloxamer USP in various grades; Hyaluronic acid; Polyacrylicacid such as carbopol 940; starch and fractionated starch; agar; alginicacid and alginates; gum Arabic; pullullan; agarose; carrageenan;dextrans; dextrin; fructans; inulin; mannans; xylans; arabinans;chitosans; glycogens; glucans; and synthetic biopolymers; as well asgums such as xanthan gum; guar gum; locust bean gum; gum Arabic;tragacanth gum; and karaya gum; and derivatives, combinations andmixtures thereof. In one embodiment of the invention, the gelling agentherein is one that is, e.g., inert to biological systems, nontoxic,simple to prepare, and/or not too runny or viscous, and will notdestabilize the IL-22 polypeptide or IL-22 Fc fusion held within it.

In certain embodiments of the invention, the polysaccharide is anetherified cellulose derivative, in another embodiment one that is welldefined, purified, and listed in USP, e.g., methylcellulose and thehydroxyalkyl cellulose derivatives, such as hydroxypropyl cellulose,hydroxyethyl cellulose, and hydroxypropyl methylcellulose (all referredto as cellulosic agents). In some embodiments, the polysaccharide ishydroxyethyl methylcellulose or hydroxypropyl methylcellulose.

The polyethylene glycol useful for gelling is typically a mixture of lowand high molecular weight polyethylene glycols to obtain the properviscosity. For example, a mixture of a polyethylene glycol of molecularweight 400-600 with one of molecular weight 1500 would be effective forthis purpose when mixed in the proper ratio to obtain a paste.

The term “water soluble” as applied to the polysaccharides andpolyethylene glycols is meant to include colloidal solutions anddispersions. In general, the solubility of the cellulose derivatives isdetermined by the degree of substitution of ether groups, and thestabilizing derivatives useful herein should have a sufficient quantityof such ether groups per anhydroglucose unit in the cellulose chain torender the derivatives water soluble. A degree of ether substitution ofat least 0.35 ether groups per anhydroglucose unit is generallysufficient. Additionally, the cellulose derivatives may be in the formof alkali metal salts, for example, the Li, Na, K, or Cs salts.

In certain embodiments, methylcellulose is employed in the gel, forexample, it comprises about 1-5%, or about 1%, about 2%, about 3%, about4% or about 5%, of the gel and the IL-22 polypeptide or IL-22 Fc fusionprotein is present in an amount of about 50-2000 μg, 100-2000 μg, or100-1000 μg per ml of gel. In certain embodiments, the effective amountof IL-22 polypeptide or IL-22 Fc fusion protein for wound healing bytopical administration can be about 25 μg to about 500 μg, about 50 μgto about 300 μg, about 100 μg to about 250 μg, about 50 μg to about 250μg, about 50 μg to about 150 μg, about 75 μg, about 100 μg, about 125μg, about 150 μg, about 175 μg, about 200 μg, about 225 μg, about 250μg, about 300 μg, or about 350 μg, per cm² wound area.

The formulations to be used for in vivo administration are generallysterile. Sterility may be readily accomplished, e.g., by filtrationthrough sterile filtration membranes.

The present invention provides dosages for the IL-22-based therapeutics.For example, depending on the type and severity of the disease, about 1μg/kg to 15 mg/kg (e.g. 0.1-20 mg/kg) of polypeptide is an initialcandidate dosage for administration to the subject, whether, forexample, by one or more separate administrations, or by continuousinfusion. A typical daily dosage might range from about 1μ.g/kg to 100mg/kg or more, depending on the factors mentioned above. For repeatedadministrations over several days or longer, depending on the condition,the treatment is sustained until a desired suppression of diseasesymptoms occurs. However, other dosage regimens can be useful. Theprogress of this therapy is easily monitored by conventional techniquesand assays.

For the prevention or treatment of disease, the appropriate dosage of apolypeptide of the invention (when used alone or in combination with oneor more other additional therapeutic agents) will depend on the type ofdisease to be treated, the type of polypeptide, the severity and courseof the disease, whether the polypeptide is administered for preventiveor therapeutic purposes, previous therapy, the subject's clinicalhistory and response to the polypeptide, and the discretion of theattending physician. The polypeptide is suitably administered to thesubject at one time or over a series of treatments. Depending on thetype and severity of the disease, about 1 μg/kg to 20 mg/kg (e.g. 0.1mg/kg-15 mg/kg) of the polypeptide can be an initial candidate dosagefor administration to the subject, whether, for example, by one or moreseparate administrations, or by continuous infusion. One typical dailydosage might range from about 1 μg/kg to 100 mg/kg or more, depending onthe factors mentioned above. For repeated administrations over severaldays or longer, depending on the condition, the treatment wouldgenerally be sustained until a desired suppression of disease symptomsoccurs. One exemplary dosage of the polypeptide would be in the rangefrom about 0.05 mg/kg to about 20 mg/kg. Thus, one or more doses ofabout 0.5 mg/kg, 2.0 mg/kg, 4.0 mg/kg, 10 mg/kg, 12 mg/kg, 15 mg/kg, or20 mg/kg (or any combination thereof) may be administered to thesubject. In certain embodiments, about 0.5 mg/kg, 1.0 mg·kg, 2.0 mg/kg,3.0 mg/kg, 4.0 mg/kg, 5.0 mg/kg, 6.0 mg/kg, 7.0 mg/kg, 8.0 mg/kg, 9.0mg/kg, 10 mg/kg, 12 mg/kg, 15 mg/kg, or 20 mg/kg (or any combinationthereof) may be administered to the subject. Such doses may beadministered intermittently, e.g. every week, every two weeks, or everythree weeks (e.g. such that the subject receives from about two to abouttwenty, or e.g. about six doses of the polypeptide). An initial higherloading dose, followed by one or more lower doses may be administered.An exemplary dosing regimen comprises administering an initial loadingdose of about 4 mg/kg, followed by a weekly maintenance dose of about 2mg/kg of the antibody. However, other dosage regimens may be useful. Theprogress of this therapy is easily monitored by conventional techniquesand assays.

The compounds of the invention for prevention or treatment of acardiovascular disease or condition, metabolic syndrome, acuteendotoxemia or sepsis, or diabetes are typically administered byintravenous injection.

Other methods of administration can also be used, which includes but isnot limited to, topical, parenteral, as intravenous, subcutaneous,intraperitoneal, intrapulmonary, intranasal, ocular, intraocular,intravitreal, intralesional, intracerobrospinal, intra-articular,intrasynovial, intrathecal, oral, or inhalation administration.Parenteral infusions include intramuscular, intravenous, intraarterial,intraperitoneal, or subcutaneous administration. In addition, thecompounds described herein are administered to a human subject, inaccord with known methods, such as intravenous administration as a bolusor by continuous infusion over a period of time.

I. Therapeutic Methods and Compositions

Any of the IL-22 Fc fusion proteins or IL-22 polypeptides or IL-22agonists provided herein may be used in therapeutic methods.

a) Inflammatory Bowel Disease

In one aspect, an IL-22 Fc fusion protein for use as a medicament isprovided. In further aspects, an IL-22 Fc fusion protein for use intreating IBD, including UC and CD, is provided. In certain embodiments,an IL-22 Fc fusion protein for use in a method of treatment is provided.In certain embodiments, the invention provides an IL-22 Fc fusionprotein for use in a method of treating an individual having UC or CDcomprising administering to the individual an effective amount of theIL-22 Fc fusion protein. In one such embodiment, the method furthercomprises administering to the individual an effective amount of atleast one additional therapeutic agent, e.g., as described below. Infurther embodiments, the invention provides an IL-22 Fc fusion proteinfor use in enhancing epithelial proliferation, differentiation and/ormigration. In certain particular embodiments, the epithelial tissue isintestinal epithelial tissue. In certain embodiments, the inventionprovides an IL-22 Fc fusion protein for use in a method of enhancingepithelial proliferation, differentiation and/or migration in anindividual comprising administering to the individual an effectiveamount of the IL-22 Fc fusion protein to enhance epithelialproliferation, differentiation and/or migration. In yet otherembodiments, the invention provides an IL-22 Fc fusion protein for usein treating diabetes, especially type II diabetes, diabetic woundhealing, metabolic syndromes and atherosclerosis. In certainembodiments, the invention provides an IL-22 Fc fusion protein for usein a method of treating diabetes, especially type II diabetes, diabeticwound healing, metabolic syndromes and atherosclerosis in an individualcomprising administering to the individual an effective amount of theIL-22 Fc fusion protein. See Genentech applications Docket numbersPR5586, application serial number 61/800,795, entitled “Using an IL-22polypeptide for wound healing,” and PR5590, application Ser. No.61/801,144, entitled “Methods of treating cardiovascular conditions andmetabolic syndrome using an IL-22 polypeptide,” both filed on Mar. 15,2013. The disclosures of both of the applications are incorporatedherein by reference in their entireties. An “individual” or “subject” or“patient” according to any of the above embodiments is preferably ahuman.

In a further aspect, the invention provides for the use of an IL-22polypeptide or IL-22 Fc fusion protein in the manufacture or preparationof a medicament. In one embodiment, the medicament is for treatment ofIBD and wound healing. In a further embodiment, the medicament is foruse in a method of treating IBD and wound healing comprisingadministering to an individual having IBD an effective amount of themedicament. In one such embodiment, the method further comprisesadministering to the individual an effective amount of at least oneadditional therapeutic agent, e.g., as described below. In a furtherembodiment, the medicament is for suppressing inflammatory response inthe gut epithelial cells. In a further embodiment, the medicament is foruse in a method of enhancing epithelial proliferation, differentiationand/or migration in an individual comprising administering to theindividual an amount effective of the medicament to enhance epithelialproliferation, differentiation and/or migration. An “individual”according to any of the above embodiments may be a human.

In a further aspect, the invention provides a method for treating IBD,including UC and CD. In one embodiment, the method comprisesadministering to an individual having IBD an effective amount of anIL-22 polypeptide or an IL-22 Fc fusion protein. In one such embodiment,the method further comprises administering to the individual aneffective amount of at least one additional therapeutic agent, asdescribed below. An “individual” according to any of the aboveembodiments may be a human.

In a further aspect, the invention provides a method for enhancingepithelial proliferation, differentiation and/or migration in anindividual. In one embodiment, the method comprises administering to theindividual an effective amount of an IL-22 polypeptide or IL-22 Fcfusion protein to enhance epithelial proliferation, differentiationand/or migration. In one embodiment, an “individual” is a human.

b) Other Therapeutic Indications

The present invention provides IL-22-based therapeutic agents forcardiovascular diseases and conditions, metabolic syndrome, acuteendotoxemia and sepsis, and diabetes. For the prevention, treatment orreduction in the severity of a given disease or condition, theappropriate dosage of a compound of the invention will depend on thetype of disease or condition to be treated, as defined above, theseverity and course of the disease or condition, whether the agent isadministered for preventive or therapeutic purposes, previous therapy,the subject's clinical history and response to the compound, and thediscretion of the attending physician. The compound is suitablyadministered to the subject at one time or over a series of treatments.Preferably, it is desirable to determine the dose-response curve and thepharmaceutical composition of the invention first in vitro, and then inuseful animal models prior to testing in humans.

In one aspect, the present invention provides methods of treatment for acardiovascular disease or disorder, metabolic syndrome, acuteendotoxemia and sepsis, and an insulin-related disorder. In oneembodiment, the method comprises administering to a subject in need atherapeutically effective amount of an IL-22 polypeptide, an IL-22 Fcfusion protein, or an IL-22 agonist. In another aspect, the inventionprovides a method for the delaying or slowing down of the progression ofa cardiovascular disease or disorder, metabolic syndrome, and aninsulin-related disorder. In one embodiment, the method comprisesadministering to subject diagnosed with the disease, condition, ordisorder, an effective amount of an IL-22 polypeptide, IL-22 Fc fusionprotein, or IL-22 agonist. In another aspect, the invention provides amethod for preventing indicia of a cardiovascular disease or disorder,and an insulin-related disorder. In one embodiment, the method comprisesadministering an effective amount of an IL-22 polypeptide, IL-22 Fcfusion protein, or IL-22 agonist to a subject at risk of the disease,condition, or disorder, wherein the IL-22 polypeptide, IL-22 Fc fusionprotein, or IL-22 agonist is effective against the development ofindicia of the disease, condition, or disorder.

Cardiovascular Diseases and Conditions

In one aspect, the IL-22 polypeptides, IL-22 Fc fusion proteins andIL-22 agonists provide a preventative or prophylactic effect against thedevelopment of, or the progression of, clinical and/or histologicaland/or biochemical and/or pathological indicia (including both symptomsand signs) of cardiovascular diseases or conditions in a subject. In oneembodiment, the disease or condition is atherosclerosis. In oneembodiment, the indicia include atherosclerotic plaque formation and/orvascular inflammation. In another embodiment, the subject is at risk forcardiovascular disease. In general, a subject at risk will previouslyhave had a cardiovascular disease or condition as described herein, orwill have a genetic predisposition for a cardiovascular disease orcondition.

The efficacy of the treatment of cardiovascular diseases and conditionscan be measured by various assessments commonly used in evaluatingcardiovascular diseases. For example, cardiovascular health can beassessed. Cardiovascular health can be evaluated by, but not limited to,e.g., blood tests (e.g., total cholesterol, LDL-C, HDL-C, triglyceride,C-reactive protein, fibrinogen, homocysteine, fasting insulin, ferritin,lipoprotein, LPS), blood pressure, auscultation, electrocardiogram,cardiac stress testing, cardiac imaging (e.g., coronary catheterization,echocardiogram, intravascular ultrasound, positron emission tomography,computed tomography angiography, and magnetic resonance imaging).

Metabolic Syndrome

In one aspect, the IL-22 polypeptides, IL-22 Fc fusion proteins andIL-22 agonists provide a therapeutic, preventative or prophylacticeffect against the development of, or the progression of, clinicaland/or histological and/or biochemical and/or pathological indicia(including both symptoms and signs) of metabolic syndrome (or metabolicdisorder or disease) in a subject. In one or more embodiment, thesubject is at risk for metabolic syndrome.

The efficacy of the treatment of metabolic syndrome can be measured byvarious assessments commonly used in evaluating metabolic syndrome. Forexample, obesity can be measured. As a further example, hyperglycemia,dyslipidemia, insulin resistance, chronic adipose tissue inflammation,and/or hypertension can be measured. Reduction in in levels of one ormore of C-reactive protein, IL-6, LPS, and plasminogen activatorinhibitor 1 can be measured. These measurements can be performed by anymethods well known in the art.

Insulin-Related Disorders

For insulin-related disorders, the term “treatment” refers to boththerapeutic treatment and prophylactic or preventative measures for thedisorder, wherein the object is to prevent or slow down (lessen) thetargeted pathologic condition or disorder. Those in need of treatmentinclude those already with an insulin-related disorder as well as thoseprone to have such a disorder or those in whom the disorder is to beprevented.

In one aspect, the IL-22 polypeptides, IL-22 Fc fusion proteins andIL-22 agonists provide a preventative or prophylactic effect against thedevelopment of, or the progression of, clinical and/or histologicaland/or biochemical and/or pathological indicia (including both symptomsand signs) of an insulin-related disorder in a subject. In oneembodiment, the disorder is Type I diabetes, Type II diabetes, orgestational diabetes. In one embodiment, the pathology or pathologicalindicia include one or more of: little or no insulin production by thepancreas (e.g., islet cells), insulin resistance, and hyperglycemia. Inanother embodiment, the subject is at risk for an insulin-relateddisorder. In general, a subject at risk has a genetic predisposition foran insulin-related disorder, has been exposed to a virus that triggersautoimmune destruction of islet cells (e.g., Epstein-Barr virus,coxsackievirus, mumps virus or cytomegalovirus), is obese, ispre-diabetic (higher than normal blood sugar levels), or has gestationaldiabetes.

The efficacy of the treatment of an insulin-related disorder can bemeasured by various assessments commonly used in evaluating suchdisorders. For example, both Type I and Type II diabetes can beevaluated with one or more of the following: a glycated hemoglobin test(A1C), a regular blood sugar test, and a fasting blood sugar test. TypeI can also be evaluated by testing for autoantibodies in the bloodand/or ketones in the urine. Type II can also be evaluated by testingfor oral glucose tolerance.

Acute Endotoxemia and Sepsis

In one aspect, the IL-22 polypeptides, IL-22 Fc fusion proteins andIL-22 agonists provide a therapeutic, preventative or prophylacticeffect against the development of, or the progression of, clinicaland/or histological and/or biochemical and/or pathological indicia(including both symptoms and signs) of acute endotoxemia, sepsis, orboth, in a subject. In one or more embodiment, the subject is at riskfor acute endotoxemia, sepsis, or both.

The efficacy of the treatment of acute endotoxemia, sepsis, or both canbe measured by various assessments commonly used in evaluating acuteendotoxemia, sepsis, or both. For example, reduction in in levels of LPSor inflammatory markers can be measured. These measurements can beperformed by any methods well known in the art.

Wound Healing

There are a variety of ways to measure wound healing. Often images aretaken to calculate linear dimensions, perimeter and area. The NIH has afree program, Image J, that allows measurement of wound areas from animage. The final healing prognosis can be extrapolated from initialhealing rates based on the migration of the periphery towards thecenter. This is done using a number of mathematical equations, the mostcommon of which is a modified Gilman's equation. In addition to visualinspection, wound healing measurement can also be aided by spectroscopicmethods or MRI. See e.g., Dargaville et al., Biosensors Bioelectronics,2013, 41:30-42, Tan et al., 2007, British J. Radiol. 80:939-48. Ifhealing is slow/inadequate, biopsies of the wound edges may be taken torule out or determine infection and malignancy. In certain embodiments,the acceleration or improvement of wound healing can be assessed bycomparing wound closure in IL-22-treated and control wounds. In certainembodiments, the acceleration or improvement of wound healing is atleast 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90% faster or better than thecontrol.

In certain aspect, the invention provides methods forpromoting/accelerating/improving healing of a wound with or withoutactive infection, microbial contamination or colonization in the wound.The IL-22 polypeptides, IL-22 Fc fusion proteins or IL-22 agonists canbe used for treating infected wounds or promoting/accelerating/improvinginfected wound healing. In certain embodiments, the IL-22 polypeptides,IL-22 Fc fusion proteins or IL-22 agonists can be used for treatingwounds, or promoting/accelerating/improving wound healing, in thepresence of infection. In some embodiments, the IL-22 polypeptides,IL-22 Fc fusion proteins or IL-22 agonists can be used for treatingwounds or promoting/accelerating/improving wound healing in the presenceof microbial contamination or colonization with risk for infection. Infurther embodiments, the patient in need of wound healing treatment canbe a diabetic patient. Accordingly, in some embodiments, the wound is adiabetic wound, for example, diabetic foot ulcer. In some furtherembodiments, the wound is an infected diabetic wound, for example,infected diabetic foot ulcer.

In a further aspect, the invention provides pharmaceutical formulationscomprising an IL-22 polypeptide, IL-22 Fc fusion protein or IL-22agonist provided herein, e.g., for use in any of the above therapeuticmethods. In one embodiment, a pharmaceutical formulation comprises anIL-22 polypeptide, IL-22 Fc fusion protein or IL-22 agonist providedherein and a pharmaceutically acceptable carrier. In another embodiment,a pharmaceutical formulation comprises an IL-22 polypeptide, IL-22 Fcfusion protein or IL-22 agonist provided herein and at least oneadditional therapeutic agent, e.g., as described below.

IL-22 Fc fusion protein of the invention can be used either alone or incombination with other agents in a therapy. For instance, an IL-22polypeptide, IL-22 Fc fusion protein or IL-22 agonist of the inventionmay be co-administered with at least one additional therapeutic agent.In certain embodiments, an additional therapeutic agent is an immunesuppressant that reduces the inflammatory response including withoutlimitation methotrexate, TNF inhibitor, TNF antagonist, mesalazine,steroid, dexamethasone, and azathioprine, and combination thereof.Suitable additional therapeutic agents that reduce an inflammatoryresponse include without limitation 5-aminosalicylic acid (5-ASA),mercaptopurine (also called 6-mercaptopurine or 6-MP) or combinationthereof. In certain embodiments, the IL22 polypeptide or IL-22 Fc fusionmay be co-administered with one or more additional therapeutic agentsthat reduce an inflammatory response (for example, 5-ASA, 6-MP, or anTNF antagonist) for the treatment of IBD. In certain other embodiments,the IL22 polypeptide or IL-22 Fc fusion may be co-administered with anintegrin antagonist such as etrolizumab for the treatment of IBD. In oneembodiment, the IL-22 polypeptide or IL-22 Fc fusion protein is used incombination with an IL-22 agonist.

For accelerating chronic wound healing, such as for the treatment ofdiabetic foot ulcer, the administration of an IL-22 polypeptide orfragments or variants thereof, IL-22 Fc fusion proteins or IL-22agonists can be combined with one or more additional wound healingagents. Suitable additional wound healing agents include withoutlimitation growth factors (e.g., EGF, FGF, IGF, PDGF, TGF, and VEGF),nerve growth factor (NGF), angiogenesis factors (e.g., HGF, TNF-α,angiogenin, IL-8, angiopoietins 1 and 2, Tie-2, integrin α5, matrixmetalloproteinases, nitric oxide, COX-2), members of the plateletderived growth factor (PDGF) family (e.g., PDGF-A, PDGF-B, PDGF-C, andPDGF-D), members of the insulin growth factor (IGF) family (e.g., IGF-I,IGF-II), members of the transforming growth factor (TGF) family (e.g.,TGF-α TGF-β) and anabolic oxygen (vacuum therapy). In certainembodiments, the IL-22 polypeptide or IL-22 Fc fusion can beco-administered with one or more additional wound healing agentsdescribed herein and/or one or more antibacterial agents or antibioticssuitable for use in topical administration. See WO2006/138468,incorporated herein by reference in its entirety. In such embodiments,the antibiotic can be sulfur antibiotic including without limitationsilver sulfadiazine, i.e., silvadeen. The co-administered one or moreadditional agents can be administered concurrently, alternatively orsequentially with IL-22 polypeptide, IL-22 fusion protein or IL22agonist.

In further exemplary embodiments, if the target is prevention ortreatment of cardiovascular diseases or conditions or metabolicsyndrome, the administration of an IL-22 polypeptide or fragments orvariants thereof, IL-22 Fc fusion proteins or IL-22 agonists can becombined with or supplement the administration of thecholesterol-lowering agents such as statins (e.g., lovastatin,rosuvastatin, fluvastatin, atorvastatin, pravastatin, and simvastatin),bile acid binding resins (colestipol, cholestyramine sucrose, andcolesevelam), ezetimibe, or a ezetimibe-simvastatin combination;anti-platelet agents such as cyclooxygenase inhibitors (aspirin),adenosine diphosphate (ADP) receptor inhibitors (clopidogrel, prasugrel,ticagrelor, ticlopidine), phosphodiesterase inhibitors (cilostazol),glycoprotein IIB/IIIA inhibitors (abciximab, eptifibatide, tirofiban),adenosine reuptake inhibitors (dipyridamole), thromboxane inhibitors(thromboxane synthase inhibitors, thromboxane receptor antagonists,terutroban); beta blockers such as alprenolol, bucindolol, carteolol,carvedilol, labetalol, nadolol, oxprenolol, penbutolol, pindolol,propranolol, sotalol, timolol, eucommia bark, acebutolol, atenolol,betaxolol, bisoprolol, celiprolol, esmolol, metoprolol, nebivolol,butaxamine, ICI-118,551, and SR 59230A; angiotensin-converting enzyme(ACE) inhibitors such as captopril, zofenopril, dicarboxylate-containingagents (enalapril, ramipril, quinapril, perindopril, lisinopril,benazepril, imidapril, zofenopril), phosphonate-containing agents(fosinopril), casokinins, lactokinins, lactotripeptides (Val-Pro-Pro,and Ile-Pro-Pro produced by the probiotic Lactobacillus helveticus orderived from casein); calcium channel blockers such as dihydropyridines(e.g., amlodipine, aranidipine, azelnidipine, barnidipine, benidipine,cilnidipine, clevidipine, isradipine, efonidipine, felodipine,lacidipine, lercanidipine, manidipine, nicardipine, nifedipine,nilvadipine, nimodipine, nisoldipine, nitrendipine, and pranidipine),phenylalkylamine (e.g., verapamil), benzothiazepines (e.g., diltiazem),mibefradil, bepridil, fluspirilene, and fendiline; diuretics such ashigh ceiling loop diuretics (e.g., furosemide, ethacrynic acid,torsemide and bumetanide), thiazides (e.g., hydrochlorothiazide acid),carbonic anhydrase inhibitors (e.g., acetazolamide and methazolamide),potassium-sparing diuretics (e.g., aldosterone antagonists:spironolactone, and epithelial sodium channel blockers: amiloride andtriamterene), and calcium-sparing diuretics, and pharmaceuticallyacceptable salts, acids or derivatives of any of the above.

For insulin-related disorders or metabolic syndrome, the administrationof an IL-22 polypeptide or fragments or variants thereof or IL-22 Fcfusion protein or IL-22 agonists can be combined with or supplement theadministration of various therapeutic agents. In the case of Type Idiabetes (insulin-dependent diabetes mellitus or IDDM), the IL-22polypeptide, Fc fusion protein or agonist described herein are combinedwith one or more of regular insulin replacement therapy (includingrapid-acting and long-acting insulin), immunosuppression treatment,islet transplantation and stem cell therapy. In one embodiment, theregular insulin replacement therapy includes, without limitation,regular insulin (e.g., Humulin R, Novolin R), insulin isophane (e.g.,Humulin N, Novolin N), insulin lispro (e.g., Humalog), insulin aspart(e.g., NovoLog), insulin glargine (e.g., Lantus) and insulin detemir(e.g., Levemir). In other embodiments, the insulin replacement therapyfurther includes pramlintide (Symlin).

In the case of Type II diabetes (non-insulin dependent diabetes mellitusor NIDDM) or metabolic syndrome, the IL-22 polypeptide, Fc fusionprotein and agonist described herein can be combined with one or more ofinsulin replacement therapy (as discussed above), an agent to lowerglucose production by the liver, an agent to stimulate pancreaticproduction and release of insulin, an agent that blocks enzymatic breakdown of carbohydrates or increases insulin sensitivity. In oneembodiment, the agent to lower glucose production is metformin (e.g.,Glucophage, Glumetza). In another embodiment, the agent to stimulatepancreatic production and release of insulin is glipizide (e.g.,Glucotrol, Glucotrol XL), glyburide (e.g., DiaBeta, Glynase) andglimepiride (e.g., Amaryl). In one other embodiment, the agent thatblocks enzymatic break down of carbohydrates or increases insulinsensitivity is pioglitazone (e.g., Actos). In another embodiment, theIL-22 polypeptide, Fc fusion protein and agonist can be combined withone of the following replacements for metformin: sitagliptin (e.g.,Januvia), saxagliptin (e.g., Onglyza), repaglinide (e.g., Prandin) andnateglinide (e.g., Starlix). Exenatide (e.g., Byetta) and liraglutide(e.g., Victoza). In another embodiment, the IL-22 polypeptide, Fc fusionprotein and agonist are combined with an oral hypoglycemic agent, e.g.,sulfonylureas.

In the case of gestational diabetes or metabolic syndrome, the IL-22polypeptide, Fc fusion and agonist described herein are combined with anoral blood sugar control medication. In one embodiment, the medicationis glyburide.

The combination therapy can provide “synergy” and prove “synergistic”,i.e. the effect achieved when the active ingredients used together isgreater than the sum of the effects that results from using thecompounds separately. A synergistic effect can be attained when theactive ingredients are: (1) co-formulated and administered or deliveredsimultaneously in a combined, unit dosage formulation; (2) delivered byalternation or in parallel as separate formulations; or (3) by someother regimen. When delivered in alternation therapy, a synergisticeffect can be attained when the compounds are administered or deliveredsequentially, e.g. by different injections in separate syringes. Ingeneral, during alternation therapy, an effective dosage of each activeingredient is administered sequentially, i.e. serially, whereas incombination therapy, effective dosages of two or more active ingredientsare administered together.

Such combination therapies noted above encompass combined administration(where two or more therapeutic agents are included in the same orseparate formulations), and separate administration, in which case,administration of the IL-22 polypeptide or IL-22 Fc fusion protein ofthe invention can occur prior to, simultaneously, and/or following,administration of the additional therapeutic agent or agents. In oneembodiment, administration of the IL-22 Fc fusion protein andadministration of an additional therapeutic agent occur within about onemonth, or within about one, two or three weeks, or within about one,two, three, four, five, or six days, of each other.

An IL-22 polypeptide or IL-22 Fc fusion protein of the invention (andany additional therapeutic agent) can be administered by any suitablemeans, including parenteral, intrapulmonary, topical and intranasal,and, if desired for local treatment, intralesional administration.Parenteral infusions include intramuscular, intravenous, intraarterial,intraperitoneal, or subcutaneous administration. Dosing can be by anysuitable route, e.g. by injections, such as intravenous or subcutaneousinjections, depending in part on whether the administration is brief orchronic. Various dosing schedules including but not limited to single ormultiple administrations over various time-points, bolus administration,and pulse infusion are contemplated herein.

IL-22 polypeptide or IL-22 Fc fusion protein of the invention would beformulated, dosed, and administered in a fashion consistent with goodmedical practice. Factors for consideration in this context include theparticular disorder being treated, the particular mammal being treated,the clinical condition of the individual patient, the cause of thedisorder, the site of delivery of the agent, the method ofadministration, the scheduling of administration, and other factorsknown to medical practitioners. The IL-22 polypeptide or IL-22 Fc fusionprotein need not be, but is optionally formulated with one or moreagents currently used to prevent or treat the disorder in question. Theeffective amount of such other agents depends on the amount of thefusion protein present in the formulation, the type of disorder ortreatment, and other factors discussed above. These are generally usedin the same dosages and with administration routes as described herein,or about from 1 to 99% of the dosages described herein, or in any dosageand by any route that is empirically/clinically determined to beappropriate.

For the prevention or treatment of disease, the appropriate dosage of anIL-22 Fc fusion protein of the invention (when used alone or incombination with one or more other additional therapeutic agents) willdepend on the type of disease to be treated, the type of Fc region, theseverity and course of the disease, whether the fusion protein isadministered for preventive or therapeutic purposes, previous therapy,the patient's clinical history and response to the IL-22 Fc fusionprotein, and the discretion of the attending physician. The IL-22 Fcfusion protein is suitably administered to the patient at one time orover a series of treatments. Depending on the type and severity of thedisease, about 1 μg/kg to 15 mg/kg (e.g. 0.1 mg/kg-10 mg/kg) or about0.1 μg/kg to 1.5 mg/kg (e.g., 0.01 mg/kg-1 mg/kg) of the IL-22 Fc fusionprotein can be an initial candidate dosage for administration to thepatient, whether, for example, by one or more separate administrations,or by continuous infusion. One typical daily dosage might range fromabout 1 μg/kg to 100 mg/kg or more, depending on the factors mentionedabove. For repeated administrations over several days or longer,depending on the condition, the treatment would generally be sustaineduntil a desired suppression of disease symptoms occurs. One exemplarydosage of the IL-22 Fc fusion protein would be in the range from about0.05 mg/kg to about 10 mg/kg. Certain other dosages include the rangefrom about 0.01 mg/kg to about 10 mg/kg, about 0.02 mg/kg to about 10mg/kg, and about 0.05 mg/kg to about 10 mg/kg. Thus, one or more dosesof about 0.01 mg/kg, 0.02 mg/kg, 0.03 mg/kg, 0.04 mg/kg, 0.05 mg/kg,0.06 mg/kg, 0.07 mg/kg, 0.08 mg/kg, 0.09 mg/kg, 0.1 mg/kg, 0.2 mg/kg,0.3 mg/kg, 0.4 mg/kg, 0.5 mg/kg, 0.6 mg/kg, 0.7 mg/kg, 0.8 mg/kg, 0.9mg/kg, 1.0 mg/kg, 2.0 mg/kg, 3.0 mg/kg, 4.0 mg/kg, 5 mg/kg, 6 mg/kg, 7mg/kg, 8 mg/kg, 9 mg/kg or 10 mg/kg (or any combination thereof) may beadministered to the patient. For topical wound healing, one or moredoses of about 0.001 mg/cm²-about 10 mg/cm² wound area, about 0.05mg/cm²-about 5 mg/cm² wound area, about 0.01 mg/cm²-about 1 mg/cm² woundarea, about 0.05 mg/cm²-about 0.5 mg/cm² wound area, about 0.01mg/cm²-about 0.5 mg/cm² wound area, about 0.05 mg/cm²-about 0.2 mg/cm²wound area, or about 0.1 mg/cm²-about 0.5 mg/cm² wound area (or anycombination thereof) may be administered to the patient. In certainembodiments, one or more doses of about 0.01 mg/cm², 0.02 mg/cm², 0.03mg/cm², 0.04 mg/cm², 0.05 mg/cm², 0.06 mg/cm², 0.07 mg/cm², 0.08 mg/cm²,0.09 mg/cm², 0.1 mg/cm², 0.15 mg/cm², 0.2 mg/cm², 0.25 mg/cm², 0.3mg/cm², 0.4 mg/cm², or 0.5 mg/cm² wound area may be administered to thepatient. Such doses may be administered intermittently, e.g. every weekor every three weeks (e.g. such that the patient receives from about twoto about twenty, or e.g. about six doses of the IL-22 Fc fusionprotein). An initial higher loading dose, followed by one or more lowerdoses may be administered. However, other dosage regimens may be useful.The progress of this therapy is easily monitored by conventionaltechniques and assays. Similar dosage ranges can be applied to an IL-22polypeptide.

It is understood that any of the above formulations or therapeuticmethods may be carried out using conjugate of the invention in place ofor in addition to an IL-22 Fc fusion protein.

J. Articles of Manufacture

In another aspect of the invention, an article of manufacture containingmaterials useful for the treatment, prevention and/or diagnosis of thedisorders described above is provided. The article of manufacturecomprises a container and a label or package insert on or associatedwith the container. Suitable containers include, for example, bottles,vials, syringes, IV solution bags, etc. The containers may be formedfrom a variety of materials such as glass or plastic. The containerholds a composition which is by itself or combined with anothercomposition effective for treating, preventing and/or diagnosing thecondition and may have a sterile access port (for example the containermay be an intravenous solution bag or a vial having a stopper pierceableby a hypodermic injection needle). At least one active agent in thecomposition is an IL-22 Fc fusion protein of the invention. The label orpackage insert indicates that the composition is used for treating thecondition of choice. Moreover, the article of manufacture may comprise(a) a first container with a composition contained therein, wherein thecomposition comprises an IL-22 Fc fusion protein of the invention; and(b) a second container with a composition contained therein, wherein thecomposition comprises a further cytotoxic or otherwise therapeuticagent. The article of manufacture in this embodiment of the inventionmay further comprise a package insert indicating that the compositionscan be used to treat a particular condition. Alternatively, oradditionally, the article of manufacture may further comprise a second(or third) container comprising a pharmaceutically-acceptable buffer,such as bacteriostatic water for injection (BWFI), phosphate-bufferedsaline, Ringer's solution and dextrose solution. It may further includeother materials desirable from a commercial and user standpoint,including other buffers, diluents, filters, needles, and syringes.

It is understood that any of the above articles of manufacture mayinclude a conjugate of the invention in place of or in addition to anIL-22 Fc fusion protein.

K. Screening Assays and Animal Models

As exemplified in the Example sections, IL-22, IL-22 Fc fusion proteinand IL-22 agonists can be evaluated in a variety of cell-based assaysand animal models of IBD, cardiovascular diseases or conditions andmetabolic syndrome.

Recombinant (transgenic) animal models can be engineered by introducingthe coding portion of the genes of interest into the genome of animalsof interest, using standard techniques for producing transgenic animals.Animals that can serve as a target for transgenic manipulation include,without limitation, mice, rats, rabbits, guinea pigs, sheep, goats,pigs, and non-human primates, e.g. baboons, chimpanzees and othermonkeys. Techniques known in the art to introduce a transgene into suchanimals include pronucleic microinjection (Hoppe and Wanger, U.S. Pat.No. 4,873,191); retrovirus-mediated gene transfer into germ lines (e.g.,Van der Putten et al., Proc. Natl. Acad. Sci. USA 82, 6148-615 [1985]);gene targeting in embryonic stem cells (Thompson et al., Cell 56,313-321 [1989]); electroporation of embryos (Lo, Mol. Cell. Biol. 3,1803-1814 [1983]); sperm-mediated gene transfer (Lavitrano et al., Cell57, 717-73 [1989]). For review, see, for example, U.S. Pat. No.4,736,866.

For the purpose of the present invention, transgenic animals includethose that carry the transgene only in part of their cells (“mosaicanimals”). The transgene can be integrated either as a single transgene,or in concatamers, e.g., head-to-head or head-to-tail tandems. Selectiveintroduction of a transgene into a particular cell type is also possibleby following, for example, the technique of Lasko et al., Proc. Natl.Acad. Sci. USA 89, 623-636 (1992).

The expression of the transgene in transgenic animals can be monitoredby standard techniques. For example, Southern blot analysis or PCRamplification can be used to verify the integration of the transgene.The level of mRNA expression can then be analyzed using techniques suchas in situ hybridization, Northern blot analysis, PCR, orimmunocytochemistry.

The animals may be further examined for signs of appropriate pathology,such as cardiovascular disease pathology, for example by histologicalexamination and/or imaging or ultrasound analysis to determineatherosclerotic plaque burden and vascular function (see Examplesbelow). Blocking experiments can also be performed in which thetransgenic animals are treated with IL-22, IL-22 Fc fusion protein or acandidate agonist to determine the extent of effects on atheroscleroticplaque formation, including the size, number, and degree of plaqueformation. In these experiments, blocking antibodies which bind to thepolypeptide of the invention are administered to the animal and thebiological effect of interest is monitored.

Alternatively, “knock out” animals can be constructed which have adefective or altered gene encoding IL-22, as a result of homologousrecombination between the endogenous gene encoding the IL-22 polypeptideand altered genomic DNA encoding the same polypeptide introduced into anembryonic cell of the animal. For example, cDNA encoding IL-22 can beused to clone genomic DNA encoding IL-22 in accordance with establishedtechniques. A portion of the genomic DNA encoding IL-22 can be deletedor replaced with another gene, such as a gene encoding a selectablemarker which can be used to monitor integration. Typically, severalkilobases of unaltered flanking DNA (both at the 5′ and 3′ ends) areincluded in the vector [see e.g., Thomas and Capecchi, Cell, 51:503(1987) for a description of homologous recombination vectors]. Thevector is introduced into an embryonic stem cell line (e.g., byelectroporation) and cells in which the introduced DNA has homologouslyrecombined with the endogenous DNA are selected [see e.g., Li et al.,Cell, 69:915 (1992)]. The selected cells are then injected into ablastocyst of an animal (e.g., a mouse or rat) to form aggregationchimeras [see e.g., Bradley, in Teratocarcinomas and Embryonic StemCells: A Practical Approach, E. J. Robertson, ed. (IRL, Oxford, 1987),pp. 113-152]. A chimeric embryo can then be implanted into a suitablepseudopregnant female foster animal and the embryo brought to term tocreate a “knock out” animal. Progeny harboring the homologouslyrecombined DNA in their germ cells can be identified by standardtechniques and used to breed animals in which all cells of the animalcontain the homologously recombined DNA. Knockout animals can becharacterized for instance, for their ability to defend against certainpathological conditions and for their development of pathologicalconditions due to absence of the IL-22 polypeptide.

Thus, the biological activity of IL-22 or its potential agonists can befurther studied in murine IL-22 knock-out mice.

The foregoing written description is considered to be sufficient toenable one skilled in the art to practice the invention. The followingExamples are offered for illustrative purposes only, and are notintended to limit the scope of the present invention in any way. Indeed,various modifications of the invention in addition to those shown anddescribed herein will become apparent to those skilled in the art fromthe foregoing description and fall within the scope of the appendedclaims.

EXAMPLES

The following are examples of methods and compositions of the invention.It is understood that various other embodiments may be practiced, giventhe general description provided above, and the examples are notintended to limit the scope of the claims.

Example 1 Cloning, Expression and Purification of the IL-22 Fc FusionProtein

General molecular cloning and protein purification techniques can beapplied in the following experiments.

i. Cloning

Full-length human IL-22 was cloned from a human colon cDNA library(Genentech). Constructs expressing human IgG1 or IgG4 IL-22Fc fusionprotein were generated for this experiment using overlapping PCRtechnique using the following primers: IL-22 Fc fusion IgG1 forwardprimer: TTGAATTCCACCATGGGATGGTCATGTATCATCCTTTTTCTAGTAGCAACTGCAACTGGAGTACATTCAGCGCCCATCAGCTCCCACTGCAGGC (SEQ ID NO:52), IL-22 Fc fusionIgG1 reverse primer AGGTCGACTCATTTACCCGGAGACAGGGAGAGG (SEQ ID NO:53),IL-22 Fc fusion IgG4 forward primer:TTGAATTCCACCATGGGATGGTCATGTATCATCCTTTTTCTAGTAGCAACTGCAACTGGAGTACATTCAGCGCCCATCAGCTCCCACTGCAGGC (SEQ ID NO:54), IL-22 Fc fusionIgG4 reverse primer: AGGTCGACTTATTTACCCAGAGACAGGGAGAGG (SEQ ID NO:55).The PCR products were cloned into expression vectors pRK5.sm(Genentech). The leader sequence (or signal peptide) was cleaved in thecell and the mature IL-22 Fc fusion did not contain the leader sequence.The clones carrying artificial linkers were cloned with primerscontaining the linker sequences. The N297G mutation was furtherintroduced by mutagenesis PCR using the following primers: IgG1 N297Gforward primer: GCG GGA GGA GCA GTA CGG AAG CAC GTA CCG TGT GG (SEQ IDNO:56), IgG1 N297G reverse primer: CCA CAC GGT ACG TGC TTC CGT ACT GCTCCT CCC GC (SEQ ID NO:57), IgG4 N297G forward primer: ACA AAG CCG CGGGAG GAG CAG TTC GGA AGC ACG TAC CGT GTG GTC AGC GTC (SEQ ID NO:58), andIgG4 N297G reverse primer: GAC GCT GAC CAC ACG GTA CGT GCT TCC GAA CTGCTC CTC CCG CGG CTT TGT (SEQ ID NO:59). Sequences of all IL-22Fcconstructs were confirmed by DNA sequencing.

ii. Cell Culture

CHO cells were grown in suspension by splitting the culture 2 times perweek to 0.3×10⁶ cells/ml in an incubator set at 37° C. and 5% CO₂.

iii. Transfection of IL-22 Fc Fusion Protein into CHO Cells and ProteinExpression

CHO cells were seeded at 1.23×10⁶ cells/ml in 720 mL culture medium. Thetransfection complex (1.6 mL PEI+800 ug DNA in 80 mL serum free media)was incubated for 10 min before added to the cells. The culture wasincubated at 33° C., 5% CO₂ for 24 hours. After further culturing for 14days. the supernatant of the culture was harvested via centrifugation.Transient CHO conditioned media (supernatant from above) was purifiedusing the MabSelect Sure (GE Healthcare) protein A affinity column. Theeluate at low pH was neutralized to pH5.0 and further purified through agel filtration column (GE Healthcare). The eluted peak was pooled,formulated and sterile filtered. The glycosylation status of the Fcregion of the fusion protein was analyzed by Mass Spectrometry asdiscussed below.

iv. Establishment of Stable Clones Expressing IL-22 Fc Fusion Protein

The plasmid encoding IL-22 Fc fusion protein was introduced into CHOcells by transfection using Lipofectamine 2000 CD (Invitrogen). Aftertransfection, the cells were centrifuged and re-plated into serum-freeselective medium. Isolates were selected for secretion of IL-22 Fc.Clones with the highest titer, as identified by ELISA, were then pooledand scaled for production.

v. Expression of IL-22 Fc Fusion Protein in E. coli

E. coli fermentation feedstock was homogenized and conditioned to 0.4%w/w PEI pH 6.7 and centrifuged. Centrate was purified using a MabSelectSure (GE Healthcare) protein A affinity column. The eluate at low pH wasneutralized to pH 5.0 and further purified through an ion exchangechromatography. Fractions were pooled, formulated and sterile filtered.

Example 2 IL-22 Fc Fusion Protein Exhibited High Percentage ofAfucosylation in the Fc Region

In this study, the glycosylation status of the Fc portion of the IL-22Fc fusion proteins was examined. Samples of purified IL-22 Fc fusionproteins from transiently transfected cells were digested with trypsin(1:25 trypsin: IL-22 Fc, w/w) for 2 hrs at 37° C. Samples were acidifiedwith trifluoroacetic acid to a final concentration of 0.1% and injectedonto a heated C18 column (PLRP-S, 1000A 8 um, Agilent) equilibrated with0.05% TFA in water. The digestion products were separated by a linearacetonitrile gradient (5 to 60%) over 20 min time. The column wasdirectly connected to the electrospray orifice of an Agilent 6520B TOFMass Spectrometer and the masses of the eluted fractions were determinedin positive ion mode. Since the Fc portions of these fusion constructsare stable in trypsin under these digestion conditions, a directcomparison of the carbohydrate status of various IL-22 fusions could bemade.

As shown in FIGS. 2A-2G, both IL-22 IgG1 and IgG4 Fc fusion proteinsshowed abnormally high levels of afucosylation. The expected masses fora glycosylated Fc of a typical monoclonal IgG1 antibody would be thoselabeled as 53296, 53458 and 53620 Da in FIG. 2A. Typically the corecarbohydrate species on each arm of the Fc would each consist of thefollowing carbohydrate composition: 4 N-acetyl glucosamine, 3 mannoseand 1 fucose sugar species (as on the peak labeled 53296 in FIG. 2A).The addition of one or two galactose sugars would produce the peakslabeled 53458 and 53620 Da, respectively (FIG. 2A). A negligible amountof molecules containing sugar moieties that was missing fucose on onearm of the Fc was detected (“−1 fucose”).

Surprisingly, human IL-22 IgG1 Fc fusion proteins of differentconstructs in which the CH2 domain is glycosylated all exhibited highlevel of afucosylation, including sugar moieties missing fucose on onearm (“−1 fucose”) and both arms of Fc (“−2 fucose”). See FIGS. 2B-2D.These afucosylated molecules comprised as high as about 30% of the totalspecies observed. Afucosylation can increase the undesirable effectoractivities of the IL-22 IgG1 Fc fusion.

IgG4 is known to have less effector function as compared to IgG1.Unexpectedly, results of Mass Spectrometry analysis also showed the “−1fucose” and “−2 fucose” glycosylated species in the trypsin-digested Fcregions of human IL-22 IgG4 Fc fusion protein. These afucosylatedmolecules comprised more than 50% of the total species observed. FIG.2E. Afucosylated antibodies have much enhanced ADCC or CDC cytotoxicityactivities, a property not desirable with these IL-22 Fc fusionproteins.

Subsequently, two additional IL-22 Fc molecules, one containing IgG1 Fcand the other IgG4 Fc were constructed in which the residue in the Fcthat would normally be glycosylated (N297) was mutated to a glycine(N297G) thereby preventing attachment of the normal core sugar. Thesewere shown to be devoid of any sugar on their Fc portions and both hadtheir expected Fc molecular weights based on their amino acid sequences(FIGS. 2F and 2G).

In summary, the Fc region of the human IL-22 Fc fusion proteins, eitherIgG1 or IgG4 Fc fusion, showed high levels of afucosylation, which canresult in increased ADCC or CDC activities, a property not desirable foruse as IL-22 therapeutics. Thus, the non-glycosylated variants weretested in further studies.

Example 3 IL-22 IgG1 and IgG4 Fc Fusion Protein in vitro Activity Assay

IL-22 engages IL-22 receptor complex and activates Jak-Stat signalingpathway. STAT3 activation is a predominant event in IL-22 mediatedsignaling pathway. In this experiment, the in vitro activities of IL-22Fc fusion proteins were measured using a luciferase reporter assay. HEK293 cells were engineered to overexpress human IL-22 receptor complexIL22R1 and IL10R2. On day 1, 1×10⁵ 293 cells were seeded in 24-wellplates in 0.4 ml Dulbecco's modified Eagle Medium (DMEM)+10% FetalBovine Serum (FBS). On day 2, cells were transfected with a STAT3-drivenluciferase reporter and a Renilla luciferase control using Lipofectamine2000 (Invitrogen) in 0.1 ml reduced serum media (Gibco Poti-MEM withreduced serum reduced by at least 50%). The STAT3 luciferase reporterconstruct contains STAT3-responsive luciferase reporter constructcontaining tandem repeats of the sis-inducible element (SIE) and thefirefly luciferase reporter gene. On day 3, IL-22 Fc fusion proteinsproduced by either transient or stable CHO clones were titrated intodifferent concentrations in 0.5 ml media, and added on top oftransfected cells. On day 4, media were removed and cells were lysedwith 100 ul passive lysis buffer (provided by the Dual-LuciferaseReporter 1000 Assay System). Twenty microliter of cell lysates weretransferred into 96-well plate and analyzed with Dual-LuciferaseReporter 1000 Assay System on luminometer (Promega). The EC50 wascalculated based on the dose-dependent activity in GraphPad Prismsoftware (La Jolla, Calif.). The EC50 values for different IL-22 Fcfusion constructs are shown in Table 2 below.

TABLE 2 IL-22 Fc Fc Constructs isotype Linker Production  EC50 (pM)  1huIgG1 DKTHT (SEQ ID NO: 32) CHO 150-200  2 huIgG1EPKSCDKTHT (SEQ ID NO: 33) CHO 350-500  3 huIgG1VEPKSCDKTHT (SEQ ID NO: 34) CHO 100-150  4 huIgG1KVEPKSCDKTHT (SEQ ID NO: 35) CHO  50-75  5 huIgG1KKVEPKSCDKTHT (SEQ ID NO: 36) CHO  25-50  6 huIgG1DKKVEPKSCDKTHT (SEQ ID CHO  25-50 NO: 37)  7 huIgG1VDKKVEPKSCDKTHT (SEQ ID CHO  25-50 NO: 38)  8 huIgG1KVDKKVEPKSCDKTHT (SEQ ID CHO 2.5-5 NO: 39)  9 huIgG1GGGDKTHT (SEQ ID NO: 41) CHO  50-75 10 huIgG1 GGGSTH (SEQ ID NO: 63) CHO 50-100 11 huIgG1 EPKSSDKTHT (SEQ ID NO: 40) CHO  50-100 12 huIgG1DKKVEPKSSDKTHT (SEQ ID CHO  25 NO: 64) 13 huIgG1KVDKKVEPKSSDKTHT (SEQ ID CHO  25 NO: 65) 14 huIgG1 DKTHT (SEQ ID NO: 32)CHO 150-200 N297A 15 huIgG1 EPKSSDKTHT (SEQ ID NO: 40) CHO  50-100 N297A16 huIgG1 DKTHT (SEQ ID NO: 32) (N297G) CHO 150-200 17 huIgG1EPKSSDKTHT (SEQ ID NO: 40) CHO  50-100 (N297G) 18 huIgG1KKVEPKSSDKTHT (SEQ ID NO: 66) CHO  20 (N297G) 19 huIgG4SKYGPP (SEQ ID NO: 43) CHO 150-200 20 huIgG4 SKYGPP (SEQ ID NO: 43) CHO 75-100 N297G 21 huIgG4 RVESKYGPP (SEQ ID NO: 44) CHO  25-50 22 huIgG4RVESKYGPP (SEQ ID NO: 44) CHO  50-75 N297G 23 huIgG1ELKTPLGDTTHT (SEQ ID NO: 42) CHO  50-75 (IgG3 linker) 24 huIgG1EPKSSDKTHT (SEQ ID NO: 40) E. coli  16 25 huIgG1-EPKSSDKTHT (SEQ ID NO: 40) E. coli  82 monomeric IL-22

A large number of IL-22 Fc fusion proteins were constructed with linkersof different length and sequences to examine the activities, stabilityand yield of each design. Linkers with native IgG sequences arepreferred to minimize potential risk of immunogenicity; however, linkerswith exogenous sequences that showed good in vitro activity wereconsidered and encompassed by the current invention.

The IL-22 IgG1 Fc fusion protein containing the DKTHT linker (SEQ IDNO:32) was tested in the STAT3 luciferase assay. See Table 2. To improveEC50 of the fusion protein, the linker length was increased from 5 to 10amino acids containing the native IgG1 sequence EPKSCDKTHT (SEQ IDNO:33). The resulting IL-22 Fc fusion protein, however, exhibitedreduced in vitro activity. See Table 2. Surprisingly, an increase in thelinker length even by one amino acid VEPKSCDKTHT (SEQ ID NO:34) improvedthe activity of the IL-22 fusion protein. Further increases in thelinker length resulted in further improvement in activity. See Table 2.

In separate experiments, the Cys in EPKSCDKTHT was changed to Ser toremove the potential of disulfide bond formation. As shown in Table 2,IL-22 Fc fusion with the linker EPKSSDKTHT (SEQ ID NO:40) showedimproved activity as compared to the parent linker sequence with the Cysresidue. Longer linker sequence incorporating the upstream sequences(into the CH1 domain of IgG1) further improved activity. Constructs withN297G mutation showed similar EC50 values when compared with the wildtype counterparts. IL-22 IgG1 (N297G) Fc fusion protein (SEQ ID NO:12)and IL-22 IgG4 (N297G) Fc fusion protein (SEQ ID NO:8) were chosen forfurther studies.

The in vitro activities of human IL-22 IgG1 (N297G) Fc fusion protein(SEQ ID NO:12) or IL-22 IgG4 (N297G) Fc fusion protein (SEQ ID NO:8)expressed from stable clones were tested in the same assay. Data in FIG.4 show representative results. Both IL-22 IgG1 and IgG4 Fc fusionproteins induced STAT3 activity at a dose-dependent manner. Both IL-22Fc fusion proteins showed similar potency. IL-22 Fc fusion proteinsexpressed from transiently transfected cells showed similar results(data not shown). As a control, native IL-22 protein produced in CHOcells was tested in the same assay, and exhibited two to three foldshigher potency than the IL-22 Fc fusion proteins.

In summary, both IgG1 and IgG4 IL-22 Fc fusion proteins exhibited invitro activity demonstrated by STAT3 luciferase assay. Further, IL-22 Fcfusion proteins with linkers of different length and sequences wereshown to activate IL-22R mediated luciferase activity.

Example 4 IL-22 Fc Fusion Proteins Reduced Symptoms of DSS-InducedColitis in Mice

Dextran Sodium Sulfate (DSS)-induced colitis is a commonly-acceptedmouse colitis model. Oral administration of DSS-containing water rapidlydamages colon epithelial cells and causes substantial body weight lossand colon epithelial structure disruption characterized by eitherimmunohistochemical (IHC) staining or histology clinical score bypathologist. In this proof of concept study, the effect of IL-22 Fcfusion protein on DSS-induced colitis was tested.

In C57BL/6 mice, colitis was induced with drinking water containing 3.5%DSS for five consecutive days starting from day 0. Mouse IL-22 IgG2a Fc(SEQ ID NO:60), a surrogate for human IL-22 Fc fusion protein was dosedthrough intraperitoneal route at 5 mg/Kg on day −1, 1, 4, and 6. Bodyweight of the animals was measured daily. On day 8, all animals weresacrificed and colon histology was studied through both IHC staining andmanual histological score.

As shown in FIGS. 5A-5C, DSS induced colitis is associated with dramaticbody weight loss (FIG. 5A), colonic epithelial damage and coloninflammation (FIG. 5B) and high histology score (FIG. 5C). IL-22Fctreatment significantly prevented weight loss, restored epithelialintegrity, diminished inflammation and reduced histology score. SeeFIGS. 5A-5C. The efficacy of IL-22 Fc exceeded the effect ofdexamethasone, the steroid standard of care (SOC) that causedsignificant body weight loss in this study.

Example 5 IL-22 Fc Fusion Protein Pharmacokinetics Study

The pilot safety and PKPD study in cynomolgus monkeys was approved bythe Institutional Animal Care and Use Committee (IACUC). The study wasconducted at Charles River Laboratories (CRL) Preclinical Services(Reno, Nev.). A total of 15 male cynomolgus monkeys (4-5 kg) from CRLstock were randomly assigned to five groups (n=3/group). Animals ingroup 1 were given an intravenous (i.v.) dose of the control vehicle onDays 1 and 8. Animals in groups 2 and 3 were given a single i.v. bolusdose of IL22-Fc IgG1 at 0.15 and 1.5 mg/kg, respectively, on Days 1 and8. Animals in groups 4 and 5 were given a single i.v. bolus dose ofIL22-Fc IgG4 at 0.15 and 1.5 mg/kg, respectively, on Days 1 and 8. Serumsamples were collected at various time points for PK and PD analysis outto Day 43 and concentrations of IL22-Fc were assessed by ELISA.

For analysis of human IL-22-Fc in cynomolgus monkey serum, mouseanti-human IL-22 mAb (Genentech) was used as a capture antibody in anELISA assay. The recombinant IL-22 Fc fusion protein was used to developa standard curve. Plate-bound IL-22-Fc was detected during a 1 hourincubation with HRP-conjugated anti-human-Fcγ-pan murine mAb (Genentech)diluted to 500 ng/mL in assay buffer. After a final wash, tetramethylbenzidine peroxidase substrate (Moss, Inc., Pasadena, Md.) was added,color was developed for 15 minutes, and the reaction was stopped with 1M phosphoric acid. The plates were read at 450 nm with a 620 nmreference using a microplate reader. The concentrations of IL-22 Fcfusion were calculated from a four-parameter fit of the IL-22 Fc fusionstandard curve.

For PK data calculations, Study Day 1 was converted to PK Day 0 toindicate the start of dose administration. All time points after the inlife dosing day are calculated as Study Day minus 1. The serumconcentration data for each animal were analyzed using 2 compartmentanalysis with WinNonlin®, Version 5.2.1 (Pharsight; Mountain View,Calif.).

The plasma concentrations of IL22-Fc showed a bi-exponential declineafter i.v. dosing (0.15 mg/kg and 1.5 mg/kg) with a short distributionphase and a long terminal elimination phase. See FIG. 6. Thetwo-compartment model with linear elimination of IL-22 Fc from thecentral compartment described the pharmacokinetic profiles for both thedoses well, suggesting negligible target mediated disposition at thesedose ranges.

The maximum serum concentration (C_(max)) andarea-under-serum-concentration-time-curve (AUC) estimated by thetwo-compartmental analysis were roughly linear and dose-proportional.See Table 3. The dose-proportional kinetics suggested IL-22R saturationat the doses tested. As shown in FIG. 6, the IL-22 IgG4 Fc fusionunexpectedly showed a 2-fold slower CL and greater than 2-fold higherexposure compared to the IgG1 Fc fusion. Without limiting to particularmechanisms, the faster clearance (CL) of IgG1 fusion may be due to lessstability of the IgG1 fusion construct because the greater than 2-foldfaster CL of the IL-22 IgG1 Fc fusion appeared to be mainly driven by alarger volume of distribution. The Beta half-lives of 4-5 days weresimilar between IgG1 and IgG4 fusions.

TABLE 3 AUC (day · C_(max) CL Beta_HL* Group μg/mL) (ug/mL) (mL/day/kg)(day) 0.15 mg/kg 4.47 ± 0.603 2.70 ± 0.607 34.0 ± 4.26 4.02 ± 0.478 IgG11.5 mg/kg 51.1 ± 9.70  30.5 ± 4.14  30.1 ± 6.18 5.33 ± 0.580 IgG1 0.15mg/kg 11.3 ± 0.752 3.99 ± 0.432  13.3 ± 0.853 4.61 ± 0.394 IgG4 1.5mg/kg 102 ± 18.9  33.4 ± 4.02  15.0 ± 2.58 5.80 ± 0.770 IgG4 *Betahalf-life

Example 6 Assessment of In Vivo Activity of IL-22Fc in Cynomolgus Monkey

Cynomolgus monkeys (Macaca fascicularis) were dosed intravenously withIL-22 Fc fusion of isotype IgG1 or IgG4 as indicated, at doses of 0.15mg/kg or 1.5 mg/kg. IL-22 binding to IL-22 receptor triggers theexpression of several genes including Serum Amyloid A (SAA),RegIII/Pancreatitis Associated Protein (PAP, also called PancrePAP), andLipopolysaccharide Binding Protein (LPS-BP). In this study, IL-22 Fcfusion protein in vivo activities were analyzed by measuring theexpression of SAA, PancrePAP, and LPS-BP. Serum samples were obtainedover a time course pre- and post-dose, as indicated in the graph.Circulating levels of monkey SAA were quantified in serum using acommercial enzyme-linked immunosorbent assay (ELISA) kit (catalog#3400-2) available from Life Diagnostics (West Chester, Pa.).Circulating levels of RegIII/PAP were quantified in serum using acommercial ELISA kit (catalog PancrePAP) produced by Dynabio (Marseille,France).

Levels of Lipoprotein Binding Protein (LBP) in serum samples weredetermined by using a qualified ELISA. Biotinylated-Lipoprotein (EnzoLife Sciences, Farmingdale, N.Y.) was coated on a Streptavidin coatedmicrotiter plate (Thermo; Rockland, Ill.). Recombinant human LBP (R&DSystems, Inc., Minneapolis, Minn.) was used as a standard in the assays.Bound LBP analyte was detected with an anti-LBP mouse monoclonalantibody (Thermo, Rockland, Ill.). Horseradish peroxidase(HRP)-conjugated F(ab′)2 fragment goat anti-mouse IgG, Fc (JacksonImmunoResearch, West Grove, Pa.) was used for detection. Thecolorimetric signals were visualized after addition of3,3′,5,5′-tetramethylbenzidine (TMB) substrate (Kirkegaard & PerryLaboratories, Gaithersburg, Md.). The reaction was stopped by additionof 1 M phosphoric acid and absorbance was measured at 450 nm using 650nm as reference on a plate reader (Molecular Devices, Sunnyvale,Calif.). All ELISA samples were run according to manufacturer'sspecifications and were prepared either at a single dilution induplicate or at four serial dilutions in singlicate and concentrationswere interpolated from a standard curve. The mean value of each samplewas reported.

As shown in FIGS. 7A-7C, SAA (FIG. 7A), LPS-BP (FIG. 7B), and RegIII/PAP(FIG. 7C) serum protein levels were induced by IL-22Fc in vivo.Dose-dependent responses were observed in vivo in non-human primates,indicating IL-22R engagement and suggesting saturation by IL-22Fc. Inthe majority of cases, no increase in the serum protein levels wasobserved 24 hours after the second dose, suggesting that serum SAA,LPS-BP, and RegIII/PAP proteins had reached the maximal levels. Serumlevels of all three proteins declined slowly over the 35-day recoveryperiod, returning to baseline in most animals. The exception being theRegIII/PAP levels in the IgG4 high dose group, which appeared to stayelevated throughout the 42-day course. This may reflect improved PK andincreased exposure by AUC for the IL-22 IgG4 Fc fusion protein ascompared to IL-22 IgG1 Fc fusion protein.

Example 7—IL-22 Treatment of Atherogenic Prone Mice (Ldlr−/−Apobec1−/−)

Recent studies have revealed IL-22's role in host defense againstpathogenic microbes. Its beneficial effects on mucosal tissuehomeostasis and immunity led us to speculate that IL-22 treatment couldalleviate endotoxemia and its pathological consequences includingatherogenic dyslipidemia, systemic inflammation and ultimately slowingthe progression of atherosclerotic disease and related disordersincluding diabetes.

To test this hypothesis atherogenic prone mice (Ldlr−/−Apobec1−/−) weretreated with an IL-22-Fc construct. These mice lack the LDL receptor andsynthesis exclusively apoB100. This model is unique in that itrecapitulates much of the pathophysiology associated with human familialhypercholesterolemia. Specifically, on a chow diet, these mice developelevated LDL cholesterol, a lipid profile with a distribution ofcholesterol similar to humans, and progressive plaque formation.Further, Ldlr−/−Apobec−/− mice have measurable risk factors thatcontribute to its cardiovascular disease, including insulin resistance,systemic inflammation, progressive plague burden, and endothelial celldysfunction. Here we demonstrate that the 3 months of treatment with theIL-22-Fc fusion protein can dramatically improve the cardiovascularhealth of these animals and reduce atherosclerotic progression.

Material and Methods

Mouse IL-22-Fc Constructs.

The IL-22-Fc construct and polypeptide used herein was typically a mouseIL-22-mouse-Fc fusion protein (SEQ ID NO:73) as shown in FIG. 32A (andDNA sequence encoding it as shown in FIG. 32B, SEQ ID NO:72). Proteinwas produced in CHO cells by transient transfections of plasmid DNA. Thefusion protein was purified by running the cell supernatant over aprotein A column followed by ion-exchange chromatography to eliminateaggregates. Serum half-life was estimated by injecting a single dose of10 mg/kg IL-22-Fc in a C57B6 mouse followed by obtaining serum from themice at specified time intervals. The serum levels of IL-22-Fc wasdetermined by a sandwich ELISA using anti IL-22 mAbs. For the in vivostudies using the Lrlr−/−Apobec1−/− double KO mice a mouse IL-22-Fcconstruct was utilized. While mouse sequences are presented and havebeen used in the examples, it is expected that in various embodimentshuman sequences can replace the mouse sequences.

Mouse Studies.

Ldlr−/−Apobec1−/− double KO mice were bred in the Genentech breedingfacility and the WT C57BL/6 mice were purchased from Jackson Laboratory.Mice were maintained in a pathogen-free animal facility at 21° C. understandard 12 hr light/12 hr dark cycle with access to chow: a standardrodent chow (Labdiet 5010, 12.7% calories from fat) or a high fat, highcarbohydrate diet (Harlan Teklad TD.03584, 58.4% calories from fat) andwater ad libitum. db/db mice in C57BLKS/J background were females andother mice used in the study were all males. The mouse IL-22-Fc orControl IgG antibody were administered through intraperitoneal (ip)route starting at the age 6 months at 50 μg/week for three months (totalof 12 weekly doses).

Analysis of Atherosclerotic Burden.

High resolution x-ray micro computed tomography was used to quantifyatherosclerotic lesion volume and atherosclerotic plaque composition.Animals were euthanized with inhalation of carbon dioxide, then perfusedvia the cardiac left ventricle with ten milliliters of phosphatebuffered saline then ten milliliters of ten percent neutral bufferedformalin. The aortas were dissected and immersed in ten percent neutralbuffered formalin for a minimum of twenty four hours and transferred toa solution of twenty percent iodine based x-ray contrast agent, Isovue370 (Bracco Diagnostics Inc., Princeton, N.J.) in ten percent neutralbuffered formalin for a minimum of twelve hours. After blotting dry, theaortas were perfused and immersed in soy bean oil (Sigma-Aldrich, St.Louis, Mo.), a low x-ray intensity background imaging media. Microcomputed tomography images were obtained using the μCT40 (Sanco Medical,Basserdorf, Switzerland) with image acquisition energy of 45 kV, acurrent of 160 μA, an integration time of 300 milliseconds with threeaverages and image resolution of twelve micrometers. The resultingimages were analyzed with Analyze (AnalyzeDirect Inc., Lenexa, Kans.) byemploying semi-automated morphological filtering and user definedregions to determine object volumes and object composition.

Assessment of Vascular Function.

Vascular function was determined by ultrasound examination of thefemoral artery to flow mediated dilatation and nitroglycerin mediateddilatation. Animals were anesthetized with two percent isoflurane, andkept at thirty seven degrees Celsius for twenty minute ultrasound exam.Nair was used to remove the hair from the ventral surface of the hindlimbs and allow for ultrasound imaging using the Vevo770 with a fiftyfive megahertz imaging probe (VisualSonics, Toronto, Canada). For flowmediated dilatation, a baseline image of the femoral artery wascollected then a rubber band was used as a temporary tunicate to occludefemoral artery blood flow for four minutes. The rubber band was thenreleased for reflow of the femoral artery and an image was acquiredevery minute for four minutes and analyzed for femoral artery maximumdiameter using manufactures supplied software tools. For nitroglycerinmediated dilatation, a baseline image of the femoral artery wascollected then an intraperitoneal injection of 20 micrograms ofnitroglycerin (Baxter, Deerfield, Ill.) was administered and an imagewas acquired every minute for four minutes and analyzed for femoralartery maximum diameter using manufactures supplied software tools.

Total Cholesterol, Triglyceride and Lipoprotein Determination.

Fresh sera samples were used to determine the total cholesterol,triglyceride, and lipoprotein distribution per manufactures instructionsusing the Cholestech LDX analysis system (Inverness Medical, Princeton,N.J.).

Sera Lipopolysaccharide Measurement.

Frozen sera samples were thawed and diluted one hundred fold inendotoxin free water and incubated at ninety degrees Celsius for tenminutes in a hot water bath. Samples were then run per manufacturesinstructions on the Endosafe-PTS system (Charles River Laboratories,Wilmington Mass.).

GTT: Glucose Tolerance Test.

The Glucose Tolerance Test (GTT) was conducted at the end of the dosingperiod with 1 g/kg i.p. glucose injection after overnight fast (14 hrs).Glucose levels were measured using One Touch Ultra glucometer. Foodconsumption was calculated during the study by individually housing themice over 4 days of acclimatizing period followed by the measurement ofone week period.

Measurement of Serum Cytokine Levels.

Serum cytokine levels were measured using Luminex 23 Multiplex panel(BioRad) through automated method. Some of the results wereindependently confirmed by Individual ELISA kits (R&D). Totalcholesterol and free fatty acids (FFA) (Roche) were determined by usingenzymatic reactions.

Results:

Ldlr−/−Apobec1−/− Mice Accurately Modeled Atherogenic Dyslipedia andwere Sensitive to Inflammatory Challenges.

The Ldlr−/−Apobec1−/− mouse model displays lipoprotein levels andextensive atherosclerotic lesions characteristic of atheroscleroticdisease in humans (Powell-Braxton et al. (1998). Nat Med 4(8): 934-8).MicroCT analysis of the aortic arch of Ldlr−/− Apobec1−/− mice revealedsigns of atherosclerotic disease as determined using an automated imageprocessing techniques on prepared samples that included the ascendingaorta, arch of the aorta, descending aorta and part of thebrachiocephalic artery. This technique also demonstrated a high degreeof heterogeneity reflecting the regional variation in severity andprogression of atherosclerosis burden that included lipid core, regionsof ruptured plaque and calcification (FIG. 8). The heterogeneity of theCT signal reflects the underlying pathology of the lesions consistentwith the complex plaque pathology of the human disease. To characterizethis model and demonstrate its sensitivity to diet inducedatherogenesis, the cohort of mice were treated with either a high fatdiet or adding fructose to their drinking water (8% w/v) for 2 months.The Ldlr−/−Apobec1−/− mice demonstrated sensitivity to these dietaryalterations with only modestly increased serum LDL but with asignificant increase in total atherosclerosis burden as compared to miceon standard chow diet (FIGS. 9A and 9B). This demonstrates that theincrease in atherosclerosis burden is likely due to inflammation ratherthan LDL increase. Further, an acute low grade inflammation stimulationwith LPS challenge (0.025 mg/Kg) resulted in a marked elevation ofproinflammatory markers in the Ldlr−/−Apobec1−/− compared with wtcontrols (FIGS. 10A-10C). The Ldlr−/−Apobec1−/− mice were also exposedto chronic LPS dosing (750 ng, ip) for 8 weeks and assessed for serumlipid profile and plaque burden. As shown in FIGS. 11A-11C, chronicendotoxin exposure results in dyslipidemia and greater plaqueinstability.

Upon treatment with IL-22-Fc, improvements in atherogenic dyslipidemiaand symptoms of metabolic syndrome were seen in the Ldlr−/−Apobec1−/−mice. These mice develop characteristics of metabolic syndrome,including insulin resistance, on a chow diet. With IL-22-Fc treatment,fasting blood glucose was reduced compared to controls and glucoseclearance was improved in the treatment group compared to control group(FIGS. 12A-12C). Thus, glucose homeostasis was improved with anormalization of glucose tolerance (GTT) and improvement in fastingglucose (FIGS. 12A-12C). Both fasted and fed hypercholesterolemia werereduced (FIG. 13A) as were fed TG levels (FIG. 13B) and the lipidprofiles were improved (FIGS. 14A-14G). Plasma LPS levels were reducedafter IL-22-Fc treatment (FIG. 15). In addition to the reduction indyslipidemia and insulin sensitization, improvement in endothelialfunction measured by vascular reactivity was seen (FIG. 16). Consistentwith an improvement in dyslipidemia, CT analysis of plaque volume showeda reduction in total atherosclerotic burden in the aortic arch and inthe brachiocephalic artery and aorta valves (FIGS. 17A-17C). Theimprovement in lipid profile and insulin resistance was not due to areduction in caloric intake since the food intake, measured over a 7 dayperiod, increased despite a modest but statistically significantreduction in body weight that occurred during the 3 months treatment(FIGS. 18A and 18B). Body weight in the control group did not changeduring the 3 month treatment protocol and the IL-22-Fc treatment groupshowed a significant reduction of body weight between the start and endof study (FIG. 18A). The average daily food intake measured over a 7 dayperiod during the course of the treatment study was elevated in theIL-22-Fc treatment group compared to control group (FIG. 18B).

Example 8—Peripheral Artery Disease Model

Stimulation of IL-22 regulated pathways by IL-22-Fc to reduceatherosclerotic progression is a potentially novel form of therapy forsubjects with cardiovascular disease and related disorder includingdiabetes and chronic kidney disease. Because cardiovascular disease,typically, is not limited to one region of a subject's vasculature, asubject who is diagnosed as having or being at risk of having coronaryartery disease is also considered at risk of developing or having otherforms of CVD such as cerebrovascular disease, aortic-iliac disease, andperipheral artery disease. The same strategy described above can be usedto validate IL-22 as a target using a mouse peripheral artery diseasemodel. The IL-22-Fc constructs are prepared and evaluated as describedabove. All necessary controls are also used. IL-22 agonists/antagonistsare evaluated and the results will validate IL-22 pathways as a targetfor drug discovery and development.

A peripheral artery disease (PAD) model based upon femoral arteryligation to create ischemic damage is used. The efficacy of the IL-22-Fcconstructs are evaluated similar to the procedures described previously(Couffinhal et al., Am. J. Pathol. 152:1667 (1998); Takeshita et al.,Lab. Invst. 75:487 (1996); Isner et al., Human Gene Therapy7:959(1996)). To test the ability of an IL-22-Fc to modulate such aperipheral arterial disease, the following experimental protocol isused: a) Using a rodent (as in the above described method), one side ofthe femoral artery is ligated to create ischemic damage to a muscle ofthe hindlimb (the other non-damaged hindlimb functions as the control);b) an IL-22-Fc polypeptide (or fragment thereof) is delivered to theanimal either intravenously and/or intramuscularly (at the damaged limb)at least 3× per week for 2-3 weeks at a range of dosages; and c) theischemic muscle tissue is collected after at 1, 2, and 3 weekspost-ligation for an analysis of biomarkers and histology. Generally,(as above) parameters for evaluation include determining viability andvascularization of tissue surrounding the ischemia, while more specificevaluation parameters may include, e.g., measuring skin blood flow, skintemperature, and factor VIII immunohistochemistry, and/or endothelialalkaline phosphatase reaction. Polypeptide expression during theischemia, is studied using any art known in situ hybridizationtechnique. Biopsy is also performed on the other side of normal muscleof the contralateral hindlimb for analysis as a control.

Alternatively, other mouse models are used (Pownall et al. US2011/0118173 A1). There are several mouse models of atherosclerosis thatwill be used to test atheroprotection. These include the apo A-I KO, apoE KO, cystathionine beta-synthase and apolipoprotein E, and the apoA-I/SR-BI double KO. These mouse models of atherosclerosis will betreated with IL-22-Fc by injection, oral dosage, or ex vivo treatment.Measurement of blood cholesterol levels after treatment with IL-22-Fcwill show an immediate decrease in total plasma cholesterol and anincreased amount of neo HDL and the subsequent appearance of matureforms of HDL, which contains cholesterol extracted from peripheraltissue over an appropriate period of hours.

Example 9—Effect of Recombinant IL-22 Fc in Diabetic Mouse Models

In our initial studies to look at the effect of IL-22-Fc in metabolicsyndromes, we noted that IL-22R KO mice were more susceptible to dietinduced obesity and insulin resistance. In subsequent experiments weobserved a loss of body fat following treatment with recombinantIL-22-Fc. In view of these data we chose to test the role of recombinantIL-22-Fc in diabetic mouse models. Efficacy end points such as fed andfasted glucose, body weight and glucose and insulin tolerance wereevaluated in this study.

Mice (10 animals/group) were treated with either Recombinant IL-22-Fc oranti Ragweed antibody as an isotype IgG control, giving 2 doses/week for3 weeks (FIG. 19):

Group 1: db/db mice (BKS.Cg-Dock7(m)+/+ Lepr(db)/J FAT): anti-Ragweedantibody (50 μg)

Group 2: db/db mice: Recombinant IL-22-Fc (50 μg)

Group 3: Diet Induced Obesity (DIO) mice: anti-Ragweed antibody (50 μg)

Group 4: Diet Induced Obesity (DIO) mice: Recombinant IL-22-Fc (50 μg)

12 week old female db/db were purchased from Jackson Laboratory and usedin the experiment. Prior to the study mice were acclimated (dailyhandling) for 7-10 days after arrival and housed single before the startof the experiment. Over days −5 to day −1 blood was collected (3-50) viatail nick for base-line glucose measurement daily. On day 0 proteinswere administered by i.p. injection (150 μl) in PBS, followed by twiceweekly doses for 3 weeks. Blood (3-5 ul) was again collected via tailnick for glucose measurement on day 2, 4, 8, 10, 14, 18 and 21. Formeasuring pK, 30 ul of blood was collected via orbital bleed underanesthesia on Days 2, 7, 13 and 20.

Recombinant IL-22-Fc or isotype IgG control antibody was dosed twice aweek through Intraperitoneal route for three weeks. The body weight andfed glucose were measured every 2 days until the end of study at day 23and glucose measurements were done through tail nick and measured usingglucometer (FIGS. 20A and 20B). In order to access the fed and fastingglucose level, on day 10 the fed glucose measurement was done in themorning and mice from both groups were fasted for 4 hours (hrs) andglucose measurements were taken using Glucometer (FIG. 20C). IL-22-Fcexposure resulted in a significant glucose lowering effect in db/dbmice.

Glucose Tolerance Test (GTT) was performed after 2 weeks of treatmentwith IL-22-Fc or IgG control at 50 μg/dose twice a week. The mice werefasted overnight (14 hrs). Fasting glucose level were measured in themorning and served as a baseline. Body weight was measured and blood wascollected (3-5 μl) via tail nick for glucose measurement. Glucosesolution at 1.5 mg/Kg body weight was administered intraperitoneally andglucose measurement was taken every 30 mins. The glucose values wererepresented in the graph for 30, 120, 180 and 220 mins. One more GTT wasperformed on day 21 following overnight fasting on day 20. Mice wereweighed daily. All the groups were euthanized on day 23 and tissues werecollected for histology. IL-22-Fc treatment demonstrated significantimprovement in glucose tolerance and insulin sensitivity (FIG. 21).

Insulin Tolerance Test (ITT) was performed after on Day 20 of the micetreated with IL-22-Fc or IgG control at 50 μg/dose twice a week. Themice were fasted for 4 hrs and baseline glucose level was taken. 1 mU/Kgbody weight was administered intraperitoneally and blood glucose levelswere monitored by tail nicks every 30 mins. In order to calculate %glucose reduction, baseline glucose level following 4 hrs fasting isnormalized to 100%. IL-22-Fc treatment was shown to significantlyimprove insulin sensitivity measured through Insulin Tolerance test(FIGS. 22A and 22B).

IL-22R is highly expressed in pancreas especially in acinar cells,although its expression status in β islet cells is still unclear. Theinsulin signal in pancreas from IL-22 Fc or control protein treateddb/db mice was examined. Histological assessment of the diabetic micewas also carried out to evaluate insulin expression in the islet cellsand the level of hepatic periportal steatosis in IL-22-Fc treatedanimals. Immunohistochemistry for insulin and glucagon was performed onformalin fixed paraffin embedded pancreas tissues as previously reported(Wu et al. 2011, Science translational medicine 3, 113ra126,doi:10.1126/scitranslmed.3002669) using rabbit anti-glucagon (CellSignaling Technologies #2760) with Alexa Fluor 555-conjugated goatanti-rabbit secondary antibody, or guinea pig anti-insulin (DAKO A0564)with Alexa Fluor 647-conjugated goat anti-guinea pig secondary antibody.The percent insulin area per islet area was calculated by dividing theinsulin positive area by the islet area minus the nuclear area.

IL-22-Fc appears to increase insulin expression in islets in db/db mice(FIG. 23A) and quantitative analysis revealed a significant increase ofboth insulin-signal intensity (FIGS. 23B, 24A, and 24B) and insulinpositive area in IL-22-Fc treated animals (FIGS. 25A and 25B), whileIL-22 Fc did not increase glucagon-signal intensity (FIG. 23C). Theinsulin positive area showed a 2.16 fold increase with IL-22-Fctreatment compared to treatment with Herceptin control (95% confidenceinterval 1.25 to 3.72). The number and area of islet were not affectedby IL-22 Fc treatment. But the β cell area per islet and the intensityof insulin staining from IL-22 Fc treated pancreas was significantlyelevated (FIGS. 23B and 52A-52C).

The pancreas beta cells of obese mice showed signs of degranulation anddegeneration (data not shown). Statistically significant higher insulinstaining was observed in beta cells of obese mice treated with IL22, ascompared to untreated obese mice (FIGS. 23A and 23B). The increase wasprobably due to increased insulin storage in the IL22 treatment group.Despite the higher level of pancreas insulin seen in IL22 treated obesemice, serum insulin levels in these mice were actually reduced ascompared to obese mice without IL22 treatment, either in fed or fastedcondition (FIGS. 23D and 23E). But the IL22 treated obese mice respondedto glucose by releasing insulin in a pattern more resembling wild typemice on chow diet, as compared to untreated obese mice (FIG. 23F). Thus,IL22 improved glucose homeostasis in obese mice potentially byincreasing granulation and improving the control mechanism of insulinrelease in the obese mice.

Next, the effect of IL-22 Fc on insulin homeostasis was examined.HFD-fed mice were treated with IL-22 Fc twice per week for 8 weeks. Theresults show that (FIGS. 23D and 23E). The data presented in FIG. 23Fshow insulin levels in mice 0 or 30 min after glucose injection. HFD-fedmice treated with IL-22 Fc, but not control HFD mice, responded toglucose injection by increasing serum insulin levels, similar to wildtype mice on Chow diet (normal diet). See FIG. 23F. Thus, IL-22 improvedglucose homeostasis in obese mice and improved insulin secretion inresponse to glucose.

As a comparison, we looked at IL-22 receptor KO mice and theirsusceptibility to diet induced obesity (DIO) and insulin resistance. TheIL-22 R KO mouse is described in FIGS. 43A-43C and below. IL-22 receptorKO mice and littermate control mice were put on 60% High Fat Diet fromweek 7 of age for 10 weeks. To assess the high fat diet (HFD) inducedglucose tolerance, mice were fasted overnight and glucose tolerance testwas performed next day morning. For this experiment, seven week oldIL-22 R KO mice and littermate age matched control animals (WT: servedas wildtype) were put on 60% HFD for 10 weeks. Mice wereintraperitoneally injected with 1.5 mg/kg body weight of glucose andblood glucose levels were monitored every 30 mins for a period of 2 hrs.Total area under curve for individual mice were calculated andgraphically represented. The data demonstrate that glucose levels aresignificantly higher in the IL-22R KO mice based on the total area underthe curve (FIGS. 27A and 27B), suggesting that the IL-22 receptor playsa role in HFD induced glucose tolerance. The IL-22 receptor KO mice didin fact put on more body weight following HFD feeding compared toLittermate WT control mice (FIG. 28).

Example 10—IL-22 Treatment of Atherogenic Prone Mice(Ldlr−/−Apobec1−/−), Resulting in Reduction in Serum LPS and SerumLDL/HDL

Nine month old Ldlr −/−, Apobec1 −/− (dko) mice were injectedintraperitoneally with 50 ug of fusion protein IL-22Fc or 50 μganti-ragweed control antibody (n=6 per group). Forty eight hours later,the animals were euthanized and serum was harvested. Lipid profiles wereanalyzed using Cholestech LDX assay, and Endotoxin was analyzed usingthe Limulus amebocyte lysate assay. Serum LPS was reduced by 50%(p=0.0052) and serum LDL/HDL was reduced by 30% (p=0.049) with IL-22-Fcas compared to anti-ragweed Fc control antibody (FIGS. 29A-29D).

In summary, mice treated with IL-22 Fc fusion protein had rapid positivechanges in lipid profile and reduction in circulating endotoxin.

Example 11 IL-22Fc Accelerated Wound Closure in Murine Diabetic WoundHealing Model, by Either Systemic or Topical Administration

Protocol

The IL-22-Fc constructs were typically a mouse IL-22-mouse-IgG2a fusionprotein (SEQ ID NOs:72 and 73) as shown in FIGS. 32A and 32B.

Mice used in the study: IL-22R KO mice and littermate control wild-type(WT) mice were bred in the Genentech animal facility. The IL-22R KO miceis described in FIGS. 43A-43C and below. The 9 weeks old Diabetic femalemice BKS.Cg-Dock7(m)+/+Lepr(db)/J FAT (db/db) andBKS.Cg-Dock7(m)+/−Lepr(db)/J lean (control BKS) were used. Mice wererandomized in the study based on body weight and fed glucose level.

The wound healing protocol was strictly followed according to IACUCRodent Survival Surgery Guidelines. Sterile technique was usedthrough-out the procedure (including sterile gloves, mask, gown, anddrape). Following induction of a surgical plane of anesthesia, thedorsal portion of the animals back (from the scapular area to the lumbararea) was shaved, stubble removed with hair remover lotion (Nair orequivalent), following rinse off with sterile water and prepped withbetadine scrub followed by alcohol rinse. The animal was placed inventral recumbency then using a 6 mm punch to mark the area of skin tobe removed (with sterile marker on the tip of the punch, then touch toskin). One 6 mm diameter full thickness skin wounds was made 1 cm leftand right of midline. The underlying perichondrium was removed withperiosteal elevator and a fine scissors.

Following this a 0.5 mm thick silicone frame, 10-12 mm inside diameter,was placed around the circular wound with superglue). Then a 2 cm squareof Tegaderm™ (3M, St. Paul, Minn.) or Opsite® (Smith & Nephew, Inc., St.Petersburg, Fla.) adhesive was placed over the wound and frame and theanimal is allowed to recover from anesthesia.

Opsite® dressings were removed every other day, wounds were inspected,treatments applied topically (20 uL of test material or saline), andfresh dressing applied. Wound gap was calculated by measuring wounddiameter from day 0 through end of the study.

In some studies fed glucose level was recorded following tail nick andusing commercial Onetouch® glucometer (lifeScan, Inc., Milpitas,Calif.).

Results

IL-22R−/− Mice Exhibited Defects in Dermal Wound Healing Response

The role of IL-22 signaling in dermal wound healing response was studiedin IL-22R KO (lacking signaling of IL-22 and its family members IL-20and IL-24). FIG. 33 shows the wound gap curve of both IL-22RKO mice(n=10) and IL-22RWT control mice (n=10) over 14 days. A 6 mm diameterwound was generated on day 0 and the gap was measured every 2 daysstaring from Day 4. Wound gap of IL-22R KO mice showed significant delayin the closure compared to WT littermate control at day 8 through day14. At the end of the study (day 14) 100% of the WT mice wounds wereclosed, compared to only 30% of mice in the IL-22RKO mice (p=0.005). Thedifferences in the wound gap between IL-22RKO and WT control mice aredeemed statistically significant at P<0.05.

Wound Healing Defect in Obese Diabetic Mice

The dermal wound healing response in diabetic condition was modeled inthe preclinical study using leptin receptor KO diabetic mice(BKS.Cg-Dock7(m)+/+Lepr(db)/J FAT) (db/db) and WT control lean mice.Circular wounds (6 mm) were generated at the back of a mouse and thewound gap closure was recorded every 2 days starting from day 4. FIGS.34A-34D show the wound gap closure (in mm) measured from day 0 throughDay 27. Throughout the study period, diabetic, obese db/db mice woundsdisplayed significant delay statistically (P<0.0001) in the woundclosure compared to Lean mice. By day 14 100% of WT mice wounds wereclosed while none of the db/db mice wounds are closed even at day 27(FIG. 35A). IL-22 expression was induced as measured by RNA levels inwild type mice days after wound excision, but not in db/db mice. SeeFIG. 35B.

IL-22Fc Accelerated Wound Closure in the Diabetic Wound Healing Model

As IL-22R−/− mice display defects in the wound closure, it washypothesized that IL-22 may influence in the wound closure. FIG. 36shows a schematic diagram of the study design. 9-week-old female obesedb/db mice were used to model diabetic wound healing. In addition toIL-22Fc (murine), anti-ragweed antibody as Fc control protein andanti-FGFR1 antibody were used as positive control. Since anti-FGFR1antibody has been demonstrated to normalize blood glucose level in thispreclinical model, it was used as a control antibody. Treatment groupswere:

-   -   Anti-Ragweed antibody (intra peritoneal (i.p.) 50 μg/dose, 8        dose)    -   IL-22Fc (intra peritoneal (i.p.) 50 μg/dose, 8 dose)    -   Anti-FGFR1 antibody (intra peritoneal (ip) 0.5 mg/kg on day 0        and day 14).        Both IL-22Fc and anti FGFR1 showed statistically significant        (P<0.001) effect in lowering glucose level in the diabetic mice        compared with anti-ragweed treatment (FIG. 37). The data        (FIG. 38) shows that systemic administration of IL-22 Fc had        striking effect in wound closure rate compared to control anti        Ragweed antibody treatment. The differences in the wound gap was        significant from starting from day 16 (P<0.05) and the wounds in        IL-22Fc treated mice was completely covered by day 27. The Fc        control antibody as well as anti FGFR1 treated mice failed to        close wounds completely even at day 27. FIGS. 39A-39E show the        wound gap measurements of individual mice at day 19, 21 and 27        where the differences in the wound gap between IL-22 Fc treated        groups compared to other 2 groups are very significant        statistically (P<0.001).

Comparison of IL-22 Fc Topical vs. Systemic Treatment

FIG. 40 shows a schematic diagram of the study design. In this study wecompared 2 modes of treatment—topical vs. systemic treatment. The groupswere:

-   -   Anti-Ragweed antibody (topical 50 μg/dose, 8 doses)    -   IL-22Fc (topical 50 ug/dose, 8 doses)    -   IL-22Fc (intra peritoneal (i.p.) 50 μg/dose, 8 doses).

The graphs in FIGS. 41A and 41B show both IL-22-Fc topical as well asIL-22-Fc systemic administration accelerated the wound closure comparedto control antibody treatment. The wound gap measurements werestatistically significantly (P≤0.001) different from day 16 through day22. No significant difference was observed with wound closure ratebetween 11-22 Fc topical and systemic treatment groups. See also FIGS.42A and 42B.

Example 12 Obese Mice Exhibited Reduced IL-22 Induction

In the following experiments, the regulation of IL-22 during immuneresponses was examined in obese mice. The major leukocyte sources ofIL-22 are innate lymphoid cells (ILCs) and T helper subsets, especiallyTh17 and Th22 cells. The IL-22 production from CD4+ T cells upon antigenchallenge in leptin receptor deficient db/db mice was examined.

Protocol

In Vivo Treatment with OVA and Flagellin.

To activate CD4 T cell in vivo, 100 μg OVA emulsified in completeFreund's adjuvant (CFA) was injected subcutaneously at lower back of theanimals, and the inguinal lymph nodes were harvested on day 7. Toactivate TLR5, 3 μg ultra-pure flagellin (InvivoGen) was injectedintravenously, and serum samples were harvested at 2 h.

Mice.

Leptin receptor deficient mice (db/db; BKS.Cg-Dock7^(m)+/+Lepr^(db)/J or

B6.BKS(D)-Lepr^(db)/J), Leptin deficient mice (ob/ob; B6.Cg-Lep^(ob)/J),and their respective lean control mice, as well as high-fat diet mice(C57BL/6J 60% DIO) and the chow-diet control mice were purchased fromJackson Laboratory. IL-22 deficient mice (Zheng et al, 2007, Nature 445,648-651) and IL-22Ra1 deficient mice (described in FIGS. 43A-43C andbelow) were generated by Lexicon Pharmaceuticals and backcrossed withC57BL/6 stain more than 10 times. Where indicated, mice were fed withadjusted calories diet (HFD, containing 60% fat, Harlan) starting at theage of 4-6 weeks old. For metabolism studies 12-18 weeks old mice wereused, whereas 5-6 weeks old mice were used for C. rodentium infectionstudies. All animal experiments were approved by the GenentechInstitutional Animal Care and Use Committee.

Naïve CD4 T Cell Purification and Differentiation.

Naïve CD4 T cells were sorted and stimulated as previously described(Rutz, et al. 2011, Nature Immunol. 12:1238-45), and cultured underspecific condition for each subset similarly to the way as describedpreviously. Id. For IL-22 induction, anti-IL-4 (10 μg/ml), anti-IFN-γ(10 μg/ml), and recombinant IL-6 (20 ng/ml) were used; where indicated,recombinant mouse leptin (1 μg/ml, R&D systems) was added.

Intracellular Staining and IL-22 ELISA.

Lymphocytes purified from draining lymph nodes were stained for IL-22and IL-17A as previously described (Zheng et al., supra) usingphycoerythrin (PE)-anti-IL-22 (1H8PWSR, eBioscience) and fluoresceinisothiocyanate (FITC)-anti-IL-17A (17B7, eBioscience). IL-22 ELISA wasperformed as previously described (Zheng et al., supra) using monoclonalanti-IL-22 antibodies (20E5 and 14B7, Genentech).

RNA Isolation and Real-Time PCR.

Colon were harvested and processed, and mRNA was isolated with RNeasymini plus kit (Qiagen). Il22, Il22ra1, and Reg3b mRNA level wereevaluated using real-time PCR analysis as previously reported (Ota etal. 2011, Nature immunol. 12, 941-948). Results were normalized to thoseof the control housekeeping gene Rp119 (encoding ribosomal protein L19)and are reported as 2^(ΔCT). The primer and probe sequence for 1122 andReg3b were reported previously. Id. For 1122ra1, 5′-AGG TCC ATT CAG ATGCTG GT-3′(SEQ ID NO:74), 5′-TAG GTG TGG TTG ACG TGG AG-3′ (SEQ ID NO:75)and 5′-FAM-CCA CCC CAC ACT CAC ACC GG-TAMRA-3′ (SEQ ID NO:76) were used.

Statistical Analysis

All statistical analysis was done with two-tailed unpaired Student'st-test. P value less than 0.05 was considered as statisticallysignificant.

Results

After immunizing the mice with ovalbumin (OVA) in Complete Freund'sAdjuvant (CFA), the IL-22 expressing CD4 T cells were detected ex vivowith intracellular cytokine staining. IL-22⁺ T cells were significantlyreduced in db/db mice (FIGS. 44A and 44B). Consistent with previousreports, IL-17+CD4 T cells were also significant reduced in db/db mice(FIG. 45A). Similar results were observed in leptin deficient ob/ob miceas well (FIG. 45B). Leptin can regulate Th cells, such as Th1 cells andTreg cells. However, a direct effect of Leptin on IL-22 production fromin vitro differentiated Th22 cells was not observed (FIG. 45C).Moreover, similar reduction of IL-22 producing T cells was also observedin immunized DIO (diet-induced obesity, or HFD-fed) C57BL/6 (FIGS. 44Cand 44D), suggesting obesity but not lack of Leptin signaling might beaccountable for the reduced IL-22 production in CD4⁺ T cells. ActivationTLR5 pathway by flagellin could stimulate IL-22 production from ILCs.

In db/db mice (FIG. 44E), ob/ob mice (FIG. 45E), and DIO mice (FIG.44F), the serum IL-22 level was significantly lower than that of WT miceupon in vivo challenge with flagellin. Consistent with the results fromT cells, leptin itself did not enhance IL-22 production from ILCs invitro (FIG. 45D). Taken together, these data suggested that there is ageneral defect in IL-22 induction from both ILCs and T cells in obesemice.

Example 13 the Mucosal Defense was Compromised in Leptin Deficient Miceand Restored by IL-22 Fc Fusion Protein

IL-22 produced by ILCs and T cells is essential for host defense againstCitrobecter rodentium infection in colon. The IL-22 induction in thecolon from db/db and ob/ob mice infected with C. rodentium was analyzed.C. rodentium was cultured overnight and mice were orally inoculated with2×10⁹ CFU of bacteria as described (Zheng et al. 2008, Nature medicine14, 282-289, doi:10.1038/nm1720). Bacterial burden was analyzed asfollows: the spleen and liver of infected mice were harvested, weighted,and homogenized in 0.1% NP40/PBS in C-tube with gentleMACS (MiltenyiBiotec). Serially diluted homogenates were plated on MacConkey agar(Remel), and C. rodentium colonies were identified as pink coloniesafter overnight incubation at 37° C. Where indicated, the mice wereinjected intramuscularly with IL-22-Fc (150 μg/dose) or equivalentamount of mouse isotype control 3 times per week. Histology analysis ofcolon from mice infected with C. rodentium was performed as reportedpreviously (Ota et al. 2011, Nature immunology 12, 941-948,doi:10.1038/ni.2089), and scored for epithelial changes (proliferation,blebbing, enterocyte shedding), inflammation, and mucosal thickening.Clinical scores were determined for four anatomic regions—proximal,middle and distal colon and rectum—on a scale from 0-5 with 0=normalcolon and 5=severe disease. Regional scores were summed to get a finalcolon disease severity score for each animal.

Corroborating with above results, the peak induction of IL-22 on day 4in the colon in db/db and ob/ob mice was also significantly reduced, butnot completely abolished (FIG. 46A). In db/db mice after oral inoculatedwith C. rodentium there was no significant weight loss (FIG. 46B).Surprisingly, the infected db/db mice started to die 10 days afterbacterial inoculation, and about 60% to 100% db/db mice succumbed duringthe second week of the infection in repeated experiments (FIG. 46C).Histological analysis of the colon sections from db/db mice revealedincreased inflammatory cell infiltration and severe epithelial damages,including epithelial shedding at the mucosal surface (FIGS. 46D-46F). Inaddition, these mice showed patchy submucosal edema and multifocalbacterial colonies, which were often associated with localized necrosis.Significantly elevated bacterial burdens were also detected in both theliver and spleen of db/db mice (FIGS. 46G and 46H). Similar defects inmucosal defense were observed in ob/ob mice as well (FIGS. 54A-54G). Itwas unexpected that db/db mice had such a significant defect incontrolling C. rodentium infection; especially given the induction IL-22by C. rodentium infection was only partially defective in these mice(see FIG. 46A).

It has been reported that Leptin deficient mice also have defects in Bcell functions, and antibody against C. rodentium is required foreventually eliminating the bacteria from the host during the later phaseof the infection. The production of anti-C. rodentium antibody in thesemice was thus examined. The serum samples were harvested by bleedingfrom submandibular vein on day 10 after the infection. ELISA plate wascoated with heat-killed C. rodentium or with a goat anti-mouse Igcapturing antibody. Coated plate was washed with washing buffer (0.05%Tween 20 in PBS), blocked for 2 h with blocking buffer (0.5% BSA, 15 ppmProclin in PBS), and washed prior to the addition of serially dilutedstandard mouse monoclonal IgG (SouthernBiotech), or serum samples. After2 h incubation at room temperature, plate was washed and the Ig weredetected with goat anti-mouse IgG conjugated with horseradish peroxidase(HRP) (SouthernBiotech), diluted 1/4,000 in assay diluent (0.5% BSA,0.05% Tween 20, 15 ppm Proclin in PBS), and incubated for 2 h at roomtemperature. After washing, TMB peroxidase substrate (Sigma-Aldrich) wasadded to each well. Absorbance was read at 650 nm in plate reader(Molecular Devices).

The titer of anti-C. rodentium IgG antibody was significantly reduced inthe survived db/db mice on day 14 after the infection (FIG. 46I).However, the reduced anti-C. rodentium IgG production alone should alsonot result in the observed early mortality, since Rag2 deficient mice,which completely lack B cells and antibody production, can survive muchlonger after infection (Zheng et al. 2008, Nature medicine 14, 282-289).Therefore, the failed host defense against C. rodentium in db/db micewere likely caused by defects in both the adaptive antibody response andthe induction of IL-22 from ILCs. Next, experiment was carried out toexamine whether IL-22 was able to restore the mucosal immunity in db/dbmice during C. rodentium infection with the administration of exogenousIL-22-Fc. As shown in FIG. 46J, while the majority of the controlIgG-treated db/db mice perished, almost all IL-22 Fc treated db/db micesurvived the infection, supporting that IL-22 Fc was able totherapeutically restore the mucosal immune defects in db/db mice.

Example 14 IL-22 Fc Reduced Glucose Levels in Obese Mice and High FatDiet-Fed Normal Mice

As described in Example 9 above, IL-22 Fc reduced glucose levels indb/db mice that already developed hyperglycemia (FIG. 20A). Thetherapeutic benefit was persistent during the course of IL-22-Fcadministration. After 3 weeks of treatment, the glucose level in thesemice dropped below 200 mg/dl, close to the normal glucose level in WTmice, while the control protein treated db/db mice sustained their highglucose level. The reduction of glucose in IL-22 Fc treated mice wasmore obvious when the mice were fasted (FIG. 20C). IL-22 Fc treatmentalso resulted in a trend of weight loss or delayed weight gain comparedto control treatment. However, at the end of this study, the weightdifference between the two groups did not reach statistical significancein these mice (FIG. 20B). Corroborating with these data, IL-22 Fctreatment led significantly improved glucose tolerance and insulinsensitivity in glucose tolerance test and insulin tolerance test (FIG.21 and FIG. 22A, respectively).

To confirm general beneficial functions of exogenous IL-22 in modulationof metabolic disorders, IL-22 Fc was administered for 4 weeks to C57BL/6mice that had been fed with HFD for at least 8 weeks to induce glucoseintolerance. For the glucose tolerance test (GTT), mice were fastedovernight, and injected i.p. with glucose solution at 1.5 mg/kg. For theinsulin tolerance test (ITT), mice were injected i.p. with insulinsolution at 1.0 unit/kg. Blood glucose was measured before and after theinjection. Blood glucose was measured by Contour (Bayer).

Consistent with the results from db/db mice, IL-22 Fc treatmentsignificantly reduced serum glucose level, especially after fasting(FIG. 47A). There was also a reduced body weight (or delayed weightgain) in the IL-22 Fc treated group at the end of the study (FIG. 47B).In addition, IL-22 Fc reduced glucose intolerance and insulin resistancein HFD-fed C57BL/6 mice (FIGS. 47C and 47D). Similar results wereobtained when mice were concurrently administrated with IL-22 Fc at thebeginning of feeding with HFD (FIGS. 48A-48E). Taken together, the datademonstrated that IL-22 Fc was a potential therapy to normalize serumglucose concentration, and alleviate glucose intolerance and insulinresistance in obese mice.

Example 15 IL-22 Fc Reduced Food Consumption and Increased Expression ofPYY in Obese and HFD-Fed Mice

The reduction of food consumption could reverse hyperglycemia andinsulin resistance in diabetic mice. Indeed, db/db mice treated withIL-22 Fc showed significant reduction of food intake in comparison withthe control group (FIG. 49A). Pair-feeding experiments were performed toensure the same food intakes in the IL-22 Fc and control treated mice(FIG. 50). Food consumption was measured for ad lib-fed group dailyduring the study. The supplied food for pair-fed group was restricted tomatch the previous day food consumption of ad lib-fed group.Correspondingly, the treatment and measurement of pair-fed group was oneday after ad lib-fed group.

Even under this condition, IL-22 Fc significantly reduced serum glucosealthough at a later time point (FIG. 49B), and reversed glucosetolerance in db/db mice (FIG. 49C), suggesting that modulating foodconsumption by IL-22 was not the only mechanism for its therapeuticeffect in metabolic disease. Similar results were observed in HFD-fedmice (data not shown). To further understand how IL-22 regulated foodconsumption and metabolism, the expression of intestine hormones, PYY,which is known to inhibit food intake was examined.

Mice were injected i.p. with 50 μg IL-22-Fc on day 0 and 2. On day 4mice were fasted overnight and re-fed for 1 h on day 5. Blood sampleswere collected on day 2 before treatment and on day 5 after feeding. Allserum samples were mixed with Protease inhibitor (Sigma), DPPIVinhibitor (Millipore) and Pefabloc (Roche) immediately after collection.PYY was measured with PYY ELISA kit (Abnova) following manufacture'sinstruction. The results show that IL-22 Fc treatment significantlyincreased PYY concentration in the serum of db/db and HFD-fed mice(FIGS. 49D and 49E). To demonstrate that IL22's effect on food intakewas mediated through promoting PYY production, food intake in micetreated with PYY inhibitor BIIE0246 was examined. C57BL/6 mice on normaldiet were either untreated or treated with IL-22 Fc on day 2 and day 4.After overnight fasting, food intake during a 4-hour feeding wasmeasured. The results show that the reduction of food intake in IL-22 Fctreated mice was reversed by BIIE0246 (data not shown), indicating thatthe effect of IL-22 Fc on reduced food intake was mediated through theinduction of PYY.

Example 16 IL-22 Fc Reduced Serum LPS and Liver ALT and AST andIncreased Lipid Metabolism in Obese Mice

Since IL-22 receptor is expressed in many organs including liver andpancreas that regulate metabolism, the therapeutic benefits of IL-22 inmetabolic diseases are likely mediated by various mechanisms. Metabolicendotoxemia contributes to inflammatory status and insulin resistanceand modulation of gut microbiota enhance glucose tolerance. Serumendotoxin was measured by Limulus Amebocyte Lysate assay kit, QCL-1000(Lonza), following manufacture's instruction. ALT and AST were measuredby Cholestech LDX (Alere). The results shown in FIG. 49F demonstratethat IL-22 Fc treatment resulted in significant reduction of the LPSamount in the serum from db/db mice.

IL-22 can repress genes involved in lipogenesis and ameliorate liversteatosis. Serum ALT and AST levels were next examined. Blood glucosewas measured by Contour (Bayer). ALT and AST were measured by CholestechLDX (Alere).As shown in FIGS. 51A and 51B, IL-22 Fc treatment loweredALT and AST levels in the serum in db/db (FIG. 51A) and HFD-fed (FIG.51B) mice. The abdominal fat was also significantly dropped with IL-22Fc treatment in HFD-fed mice (FIG. 51C). In addition, genes responsiblefor lipid metabolism were induced by IL-22 in primary adipocytes (FIG.51D). Next, the effect of IL-22 on triglyceride and cholesterol in liverand adipose tissue were examined. The results show that IL-22 Fc reducedtriglyceride, cholesterol, and free fatty acid (FFA) (FIG. 51E), as wellas hepatic triglyceride (FIG. 51F), hepatic cholesterol (FIG. 51G) andtriglyceride in white adipose tissue (FIG. 51H) in HFD-fed mice.Similarly, IL-22 reduced triglyceride in the liver and white adiposetissue in db/db mice (FIGS. 51I and 51J). Further experiments show thatIL-22 Fc treatment reduced inflammatory cytokines such as TNFα and IL-1βas compared to no treatment in obese mice (data not shown). H&E stainingof liver sections revealed a decrease in hepatic periportal steatosiswith IL-22 Fc fusion protein treatment (FIGS. 26A and 26B).

IL-22 signals through IL-22R1 and IL-10R2 chains. IL-22R1 can also bepaired with IL-20R2 chain and be utilized by IL-20 and IL-24. It hasbeen shown that all these ligands induced very similar downstreambiological effects from skin epidermis (Sa et al., 2007, J Immunol 178,2229-2240). Thus, both the IL-22 and IL-22R1 deficient mice wereexamined to avoid potential redundancy of other cytokines in HFD induceddiabetes. The generation of IL-22R knock out mice is illustrated in FIG.43A. The deletion of IL-22R1 in the KO mice was confirmed by the absenceof IL-22R1 mRNA in the IL-22R KO mice, and the lack of RegMb mRNAexpression in response to IL-22 Fc in the IL-22R KO mice. See FIGS. 43Band 43C. In addition, administration of IL-22-Fc to IL-22R KO mice didnot induce pStat3 (data not shown).

No difference was observed in glucose tolerance and body weight in IL-22deficient mice from those of WT littermate controls (FIG. 53). WhenIL-22R1 deficient mice were treated with high fat diets for threemonths, however, these mice developed significantly more severe glucosetolerance and gained more weight (FIGS. 49G-49I), supporting a criticalrole of IL-22R pathway in controlling metabolism. The possibility ofIL-20 and IL-24 redundancy in reducing metabolic syndrome was examined.In this experiment, db/db mice were treated with IL-20 Fc, IL-22 Fc orIL-24 Fc. The result indicates that only IL-22 Fc reduced serum glucoselevel (FIG. 55B) and improved glucose tolerance in a GTT assay on day 20(FIG. 55C) in db/db mice, while treatment of db/db mice with IL-20 Fc orIL-24 Fc did not. The reduction of body weight was not statisticallysignificant. Further experiments show that although IL-20 Fc and IL-24induced pStat3 in primary adipocytes, these cytokines failed to inducepStat3 in liver tissue from db/db mice that had become insensitive toinsulin (data not shown). Treatment of IL-22 Fc in the IL-22R KO micehad no effect in a glucose tolerance test, confirming that the effect ofIL-22 Fc was exerted through the IL-22 R signaling (data not shown).

The studies presented here indicate critical functions of IL-22 inregulating metabolic processes. IL-22R1 deficient mice were predisposedto development of metabolic syndromes. Exogenous IL-22 was not only ableto restore the mucosal immune defects in preclinical diabetic models,but also helped to normalize glucose and lipid metabolisms. IL-22, thus,can provide a novel therapeutic approach to treat human metabolicdisorders.

Example 17 Comparison of VGEF and IL-22 in Promoting Wound Healing indb/db Mice

In this experiment, the effect of IL-22 on promoting or improving woundhealing was analyzed and compared with that of VEGF. FemaleBKS.Cg-Dock7^(m)+/+Lepr^(db)/J db/db mice of 11 weeks of age werepurchased from Jackson Laboratory, Bar Harbor, Me. All experimentalanimal studies were conducted under the approval of the InstitutionalAnimal Care and Use Committees of Genentech Lab Animal Research. Underisoflurane anesthesia the dorsal skin was shaved then depilatory creamwas applied to remove the remaining stubble. After the skin is cleanedand prepped with povidone-iodine followed by alcohol swabs, a circular,full-thickness wound was created on the dorsal skin of each mouse usinga disposable 6 mm biopsy punch (Miltex, Inc.). The wound was coveredwith a Tegaderm film before and after treatment.

The results in FIGS. 56A and 56B show that VEGF appeared to achievefaster surface closure as compared with IL-22; however, when the dermisside of the skin was examined, wounds treated with VEGF remained openeven on day 21 (FIG. 56B). The ability of VEGF and IL-22 Fc in promotingangiogenesis at the wound site was also analyzed. In this experiment,two 6 mm wounds were excised in db/db mice on day 0. On day 2, 4, 6, 8,10 and 12, either control anti-ragweed antibody or IL-22 Fc (50 μg) orVEGF (20 μg) in saline was administered topically onto the wounds. Onday 6 and 12, three mice from each group were taken down for histologyand immunohistochemistry analysis and BrdU staining. On day 16, onemouse was taken down for BrdU staining. On day 18, 20 and 22, one mousefrom each group was taken down for each time point forimmunohistochemistry analysis and CD31 whole tissue staining. Theresults indicate that both VEGF and IL22-Fc, but not the controlanti-ragweed antibody, promoted blood vessel formation at the wound siteas analyzed by CD31 tissue immunostaining (data not shown).

Next, we analyzed IL-22-induced and other IL-10 family member-inducedcytokine and chemokine expression in reconstituted epidermis. Thereconstituted epidermis was EpiDerm RHE tissue models maintained inEPI-100-NMM medium purchased from MatTek. See Sa et al. 2007, J.Immunol. 178:2229-2240. The results show that IL-22 prominently inducedexpression of IL-8, CXCL-1, MIP 3a, DMC, and MCP-1 in reconstitutedhuman epidermis, though inductions by IL-19, IL-20 or IL-24 were alsoobserved (FIGS. 57A-57E). In view of the effect of IL-22 on woundhealing described herein, IL-19, IL-20, and IL-24 may also play a rolein accelerating wound healing.

Example 18 IL-22 Provides Superior Efficacy in the Treatment of InfectedWound than VEGF and PDGF in a Splinted Wound Model in db/db Mice

In the mouse wound healing model, contraction accounts for a large partof wound closure in mice because mice skin is mobile. To more closelyresemble the wound healing process in human, a mouse splinted woundmodel was established in which a silicon ring was glued to the skin andanchored with sutures around the wound to prevent local skin contraction(see representative images in FIG. 59B). See e.g., Zhao et al., 2012,Wound Rep. Reg. 20:342-352 and Brubaker et al., 2013, J. Immunol.,190:1746-57. In this model, wounds healed through granulation andre-epithelialization processes, similar to the wound healing processesin humans. To splint the wound, Krazy glue (Elmer's Products, Inc.) wasapplied to one side of the sterile silicone splint (Grace Bio-Labs,Inc.) and the splint was carefully placed around the wound with the glueside down so that the wound was centered within the splint. The gluebonded to the skin on contact and served as a splint for the entirecourse of the study. The splint was further anchored to the skin withfour interrupted 6.0 monofilament nylon suture (Ethicon, Inc.). Digitalimage of the wound was taken before the wound was covered with aTegaderm transparent film. Further, microbial infection on the openwound can delay wound healing, and chronic wounds, such as chronicwounds observed in diabetic patients, are often infected wounds.

Using the splinted wound model, the effect of IL-22 Fc on infected woundwas examined in db/db mice. Wounds excised as described above in wildtype or db/db mice were inoculated topically with 0.5×10⁶ CFU, 1×10⁶ CFU(plaque forming unit) or 2×10⁶ CFU of Staphylococcus aureus two daysafter wound excision. As shown in FIG. 58, db/db mice exhibited delayedwound healing as compared to wild type mice, and wound healing wasfurther delayed when the wound was infected by bacteria in these mice ascompared to control.

In separate experiments, IL-22's wound healing effect was compared withother agents in the splinted infected wound model. Two days after woundexcision, the methicillin-resistant S. aureus strain USA300 NRS 384(NARSA) at 1×10⁶ CFU in 30 ul saline was inoculated onto the woundsurface and covered again with a Tegaderm film. Topical treatment began48 hours after S. aureus infection with 30 ug of either IL22-Fc or VEGF(Lot #110308, Genentech) or PDGF (Lot #0507CY420, PeproTech, Inc.) in 30ul of saline 3 times a week thereafter. Digital images of the wound wererecorded before treatment and twice a week after treatment until closureof the wounds. Percentage of wound closure was calculated from the woundimages using ImageJ, a java-based image processing program developed atthe NIH.

As shown in FIGS. 59A and 59B, IL-22 Fc promoted faster wound healingthan VEGF when same amount of the compounds was applied to infectedwounds in the splinted wound model, which more closely resembled woundhealing in human. Next, different doses of VEGF and IL-22 Fc were testedon infected wounds. In this experiment, one 6 mm diameter splintedexcisional wound was created in db/db mice with blood glucose >300mg/dl. At each wound 1×10⁶ CFU of S. aureus USA 300 was inoculated.Varying doses of VEGF or IL-22 Fc in saline were administered topicallythree times per week until wound closure. Saline was used as a control.At wound closure, mice were sacrificed and samples were subjected tohistology, immune-histochemistry, and PCR analysis and CFU count. Theresults in FIG. 60 show that IL-22 Fc at the amount of 30 μgdemonstrated better infected wound healing efficacy than 60 μg VEGF.Thus, the faster surface closure by VEGF observed in a non-splintedwound model was likely due to mouse skin contraction and the effects ofIL-22 Fc on promoting keratinocyte proliferation and re-epithelializatinwere likely masked by mouse skin contraction.

Similar results are shown in FIG. 61, in which IL-22 Fc was demonstratedof having superior efficacy than VEGF and PDGF when the same amount (30μg) of each compound was applied to the wound. Complete wound closure inIL-22 Fc treated infected splinted wound was seen on day 15. In VEGF- orPDGF-treated mice, however, complete closure of infected splinted woundwas not seen until day 25, same as untreated uninfected wound. Woundclosure in the control group, i.e., untreated infected wound, was notseen until day 29. Without being limited to specific mechanism(s), thesuperiority of IL-22 Fc in promoting wound healing than VEGF or PDGF canbe due to its effects on re-epithelialization, promoting keratinocyteproliferation, induction of neovascularization, induction of proteasesto facilitate tissue remodeling and repair and the antimicrobialactivities.

Next, we tested whether IL-22 Fc can be administered in a gelformulation for wound healing. The exemplary gel formulation used inthis experiment contained 10 mM sodium phosphate at pH 7.1 with 0.5 mg/gMethionine and 3% Hydroxypropyl methylcellulose (HPMC E4M premium fromDow Chemicals), with or without 1 mg/g IL-22 Fc. The gel solution andIL-22 Fc solution were mixed prior to being applied topically to thesplinted wound. The formulation containing IL-22 Fc also contained asmall amount of sucrose (<20 mM) and P20 (<0.002%) carried from theoriginal protein formulation. The results shown in FIG. 62 demonstratethat IL-22 Fc in both solution and gel formulation promoted woundhealing in a non-infected splinted wound.

The specification is considered to be sufficient to enable one skilledin the art to practice the invention. Although the foregoing inventionhas been described in some detail by way of illustration and example forpurposes of clarity of understanding, the descriptions and examplesshould not be construed as limiting the scope of the invention. Indeed,various modifications of the invention in addition to those shown anddescribed herein will become apparent to those skilled in the art fromthe foregoing description and fall within the scope of the appendedclaims.

What is claimed is:
 1. A method for treating a cardiovascular conditionin a subject, which condition includes a pathology of atheroscleroticplaque formation, the method comprising administering to a subject inneed thereof a therapeutically effective amount of an IL-22 Fc fusionprotein, wherein the IL-22 Fc fusion protein binds to IL-22 receptor andcomprises an IL-22 polypeptide linked by a linker to an IgG4 Fc regionthat is not glycosylated, wherein the IL-22 Fc fusion protein comprisesan amino acid sequence having at least 98% sequence identity to theamino acid sequence of SEQ ID NO:8, and wherein the linker consists ofthe amino acid sequence of RVESKYGPP (SEQ ID NO:44).
 2. The method ofclaim 1, wherein the cardiovascular condition is selected from the groupconsisting of coronary artery disease, coronary microvascular disease,stroke, carotid artery disease, peripheral arterial disease, and chronickidney disease.
 3. The method of claim 1, wherein the subject is human.4. The method of claim 1, wherein the Fc region comprises an alteredglycosylation consensus site.
 5. The method of claim 1, wherein the Fcregion comprises an amino acid insertion, deletion, or substitution thatresults in an aglycosylated Fc region.
 6. The method of claim 1, whereinin the Fc region the amino acid residue at position 297 as in the EUindex is substituted and/or the amino acid residue at position 299 as inthe EU index is substituted.
 7. The method of claim 6, wherein the aminoacid residue at position 297 as in the EU index is Gly, Ala, Gln, Asp,or Glu.
 8. The method of claim 7, wherein the amino acid residue atposition 297 as in the EU index is Gly or Ala.
 9. The method of claim 8,wherein the amino acid residue at position 297 as in the EU index isGly.
 10. The method of claim 6, wherein the amino acid residue atposition 299 as in the EU index is Ala, Gly, or Val.
 11. The method ofclaim 1, wherein the IL-22 polypeptide is a human IL-22 polypeptide. 12.The method of claim 1, wherein the IL-22 polypeptide comprises the aminoacid sequence of SEQ ID NO:4.
 13. The method of claim 1, wherein theIL-22 receptor is a human IL-22 receptor.
 14. The method of claim 1,wherein the amino acid sequence has at least 99% sequence identity tothe amino acid sequence of SEQ ID NO:8.
 15. The method of claim 1,wherein the IL-22 Fc fusion protein comprises the amino acid sequence ofSEQ ID NO:8.
 16. The method of claim 1, wherein the IL-22 Fc fusionprotein comprises the amino acid sequence of SEQ ID NO:10.
 17. Themethod of claim 1, wherein the IL-22 Fc fusion protein comprises theamino acid sequence of SEQ ID NO:16.
 18. The method of claim 1, whereinthe IL-22 Fc fusion protein consists of the amino acid sequence of SEQID NO:8, SEQ ID NO:10, or SEQ ID NO:16.
 19. The method of claim 1,wherein the IL-22 Fc fusion protein is produced by a process comprisingthe step of culturing a host cell capable of expressing the IL-22 Fcfusion protein under conditions suitable for expression of the IL-22 Fcfusion protein.
 20. The method of claim 19, wherein the process furthercomprises the step of obtaining the IL-22 Fc fusion protein from thecell culture or culture medium.
 21. The method of claim 19, wherein thehost cell is a Chinese hamster ovary (CHO) cell.
 22. The method of claim1, wherein the IL-22 Fc fusion protein is a dimeric IL-22 Fc fusionprotein.
 23. The method of claim 1, wherein the IL-22 Fc fusion proteinis a monomeric IL-22 Fc fusion protein.
 24. The method of claim 1,wherein the IL-22 Fc fusion protein is administered intravenously,subcutaneously, or intraperitoneally.
 25. The method of claim 1, whereinthe subject is co-administered at least one additional therapeuticagent.
 26. The method of claim 1, wherein the IL-22 Fc fusion protein isadministered in a pharmaceutical composition comprising the IL-22 Fcfusion protein and at least one pharmaceutically acceptable carrier. 27.The method of claim 26, wherein the pharmaceutical composition isadministered intravenously, subcutaneously, or intraperitoneally. 28.The method of claim 26, wherein the pharmaceutical composition comprisesan additional therapeutic agent.